Use of mycrobacterial vaccines in CD4+ or CD8+ lymphocyte-deficient mammals

ABSTRACT

Methods of treating a mammal that is deficient in CD4 +  and/or CD8 +  lymphocytes are provided. The methods comprise inoculating the mammal with an attenuated mycobacterium in the  M. tuberculosis  complex. In these methods, the mycobacterium comprises two deletions, wherein a virulent mycobacterium in the  M. tuberculosis  complex having either deletion exhibits attenuated virulence. Use of these mycobacteria for the manufacture of a medicament for the treatment of mammals deficient in CD4 +  and/or CD8 +  lymphocytes is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/US2004/001773, filed Jan. 23, 2004, which claims the benefit of U.S.Provisional Application No. 60/442,631, filed Jan. 24, 2003.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided by the terms of AI26170 awardedby National Institutes of Health.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention generally relates to live bacterial vaccines. Morespecifically, the invention is related to novel Mycobacterium sp.compositions, and the use of those compositions to protect mammalsagainst disease caused by virulent Mycobacterium sp.

(2) Description of the Related Art

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There exists an urgent need for a novel tuberculosis (TB) vaccine asthere are more than 8 million new cases of tuberculosis and more than 2million deaths reported each year by the WHO (Dye et al., 1999). Thediscovery of the causative agent of TB, Mycobacterium tuberculosis, byRobert Koch in 1882 opened up the possibility for a novel vaccine (Koch,1882). Since then, numerous attempts to develop attenuated vaccinesagainst tuberculosis have failed, including tuberculin (a proteinextract of killed tubercle bacilli) developed by Dr. Koch himself. Thisfailure of tuberculin to protect led to a “firm conviction that immunitycould only be established by inducing a definite, albeit limited,tuberculosis process” Grange et al., 1983). Thus, numerous labs set outto follow the example of Dr. Louis Pasteur for viruses and enrichattenuated mutants of the tubercle bacillus following repeatedpassaging.

In order to test the hypothesis that a tubercle bacillus isolated fromcattle (now known as M. bovis) could transmit pulmonary tuberculosisfollowing oral administration, Drs. Calmette and Guerin developed amedium containing beef bile that enabled the preparation of finehomogenous bacillary suspensions (Calmette and Guerin, 1905). An M.bovis strain obtained from Dr. Norcard, was passaged every 21 days inthis medium and after the 39th passage, the strain was found to beunable to kill experimental animals (Gheorghiu, 1996). “Between 1908 and1921, the strain showed no reversion to virulence after 230 passages onbile potato medium” (Id.), which is consistent with the attenuatingmutation being a deletion mutation. In the animal studies that followed,the strain (‘BCG’) was found to be attenuated but it also protectedanimals receiving a lethal challenge of virulent tubercle bacilli(Calmette and Guerin, 1920). BCG was first used as a vaccine againsttuberculosis in 1921. From 1921 to 1927, BCG was shown to haveprotective efficacy against TB in a study on children (Weill-Halle andTurpin, 1925; Calmette and Plotz, 1929) and adopted by the League ofNations in 1928 for widespread use in the prevention of tuberculosis. Bythe 1950's after a series of clinical trials, the WHO was encouragingwidespread use of BCG vaccine throughout the world (Fine and Rodrigues,1990). Although an estimated 3 billion doses have been used to vaccinatethe human population against tuberculosis, the mechanism that causesBCG's attenuation remains unknown.

Mahairas et al. (1996) first compared the genomic sequences of BCG andM. bovis using subtractive hybridization and found that there were threemajor deletions (named RD1, RD2, and RD3) present in the genome of M.bovis, but missing in BCG. Behr et al. (1999) and others (Gordon et al.,2001) later identified 16 large deletions, including RD1 to RD3, presentin the BCG genome but absent in M. tuberculosis. These authors concludedthat 11 of these 16 deletions were unique to M. bovis, while theremaining 5 deletions were unique to BCG. They also found that one ofthese 5 deletions, designated RD1 (9454 bp), is present in all of theBCG substrains currently used as TB vaccines worldwide and concludedthat the deletion of RD1 appeared to have occurred very early during thedevelopment of BCG, probably prior to 1921 (Behr et al., 1999).

The development of insertional mutagenesis systems for BCG and M.tuberculosis (Kalpana et al., 1991), transposon mutagenesis systems(Cirillo et al., 1991; McAdam et al., 1995; Bardarov et al., 1997) andallelic exchange systems (Balasubramanian et al., 1996; Pelicic et al.,1997) led to the isolation of the first auxotrophic (nutrient-requiring)mutants of these slow-growing mycobacteria. Auxotrophic mutants of BCGand M. tuberculosis have been shown to confer protection to M.tuberculosis challenges with variable efficacies (Guleria et al., 1996;Smith et al., 2001). However, a head-to-head comparison of BCG to aleucine auxotroph of BCG showed that a single immunization elicited nopositive skin-test and imparted little immunity to challenges with M.tuberculosis or M. bovis (Chambers et al., 2000). In contrast, amethionine auxotroph of BCG that grows in vivo did confer significantprotection to challenge to both M. tuberculosis and M. bovis (Id.). Asingle dose of a leucine auxotroph of M. tuberculosis failed to elicitprotection as good as BCG in BALB/c mice (Hondalus et al., 2000). Theseresults suggest that optimal immunity against M. tuberculosis requiressome growth of the immunizing strain. Double mutants of M. tuberculosishave also been created (Parish and Stoker, 2000), but whether suchmutants are improved over single attenuating mutants in protectingmammals against challenge with a virulent mycobacterium, particularlywhen the host is immunocompromised, has not been established.

It is also worth noting that in the study of Chambers et al. (2000),both BCG and the BCG mutants seemed to protect better against M. bovischallenge than M. tuberculosis. If we assume the reverse correlate istrue, we could hypothesize that optimal immunity against M. tuberculosiscould be achieved with a M. tuberculosis-derived mutant that grew in themammalian host.

Based on the above, there remains a need for improved live mycobacterialvaccines having attenuated virulence, that confer protection fromvirulent mycobacteria, particularly M. tuberculosis. The need isparticularly acute for immunocompromised individuals. The instantinvention satisfies that need.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that mycobacteria havingtwo attenuating mutations are safe and protect mammals that aredeficient in CD4⁺ and/or CD8⁺ lymphocytes from challenge by virulentmycobacteria.

Thus, the present invention is directed to methods of treating a mammalthat does not have severe combined immune deficiency but is deficient inCD4⁺ lymphocytes. The methods comprise inoculating the mammal with anattenuated mycobacterium in the Mycobacterium tuberculosis (M.tuberculosis) complex. In these embodiments, the mycobacterium comprisestwo deletions, where a virulent mycobacterium in the M. tuberculosiscomplex having either deletion exhibits attenuated virulence.

The invention is also directed to methods of treating a mammal that doesnot have severe combined immune deficiency but is deficient in CD8⁺lymphocytes. The methods comprise inoculating the mammal with anattenuated mycobacterium in the Mycobacterium tuberculosis (M.tuberculosis) complex. In these embodiments, the mycobacterium comprisestwo deletions, where a virulent mycobacterium in the M. tuberculosiscomplex having either deletion exhibits attenuated virulence.

Additionally, the invention is directed to the use of an attenuatedmycobacterium in the Mycobacterium tuberculosis (M. tuberculosis)complex for the manufacture of a medicament for treatment of a mammalthat is that does not have severe combined immune deficiency but isdeficient in CD4⁺ lymphocytes. In these embodiments, the mycobacteriumcomprises two deletions, where a virulent mycobacterium in the M.tuberculosis complex having either deletion exhibits attenuatedvirulence.

In other embodiments, the invention is directed to the use of anattenuated mycobacterium in the Mycobacterium tuberculosis (M.tuberculosis) complex for the manufacture of a medicament for treatmentof a mammal that does not have severe combined immune deficiency but isdeficient in CD8⁺ lymphocytes. In these embodiments, the mycobacteriumcomprises two deletions, where a virulent mycobacterium in the M.tuberculosis complex having either deletion exhibits attenuatedvirulence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows maps and autoradiographs pertaining to the construction ofΔRD1 mutants of M. tuberculosis. Panel a, M. tuberculosis H37Rvpublished sequence between 4346 kb and 4364 kb, showing predicted NcoIsites. Arrows on the top represent the genes in the RD1 region. The RD1region deleted from M. bovis BCG is represented by an open bar spanningfrom Rv3871 to Rv3879c. Upstream and downstream flanking sequences, UFSand DFS respectively, are indicated as closed bars underneath the gridline. Panel b, Southern hybridization of M. tuberculosis H37Rv ΔRD1created using two-step sequential homologous recombination. Panel c,Southern hybridization of M. tuberculosis H37Rv and Erdman ΔRD1 strainscreated using specialized transduction.

FIG. 2 shows graphs summarizing experiments establishing that M.tuberculosis H37Rv ΔRD1 is attenuated in SCID mice. Panel a, Sevenfemale SCID mice were infected intravenously with 2×10⁶ CFU M.tuberculosis H37Rv, M. tuberculosis H37Rv ΔRD1, and M. tuberculosisH37Rv ΔRD1::2F9 per mouse. The number of surviving mice was recordedpost infection. Panel b, Mice were infected with different doses of M.tuberculosis H37Rv, M. tuberculosis H37Rv ΔRD1, and M. bovis BCG. Foreach strain, infection doses of 2×10⁶CFU, 2×10⁵ CFU, 2×10⁴ CFU, and2×10³ CFU per mouse, were administered via tail intravenous injection.

FIG. 3 a-3 g are photographs, micrographs and autoradiographs showingthat the M. tuberculosis H37Rv ARD1 mutant exhibits two distinctcolonial morphotypes. Panel a, M. tuberculosis H37Rv. Panel b, M.tuberculosis H37Rv ΔRD1. Panel c, M. tuberculosis H37Rv ΔRD1::2F9. Paneld, Southern analysis of M. tuberculosis H37Rv ΔRD1 NcoI-digested genomicDNA, isolated from three smooth and three rough colonies and probed withDFS. Panels e-g, Colonial morphotypes at higher magnification. e, Smoothmorphotype at week 4. f, Rough morphotype at week 4. g, Rough morphotypeat week 6.

FIG. 4 a-4 d are graphs showing the growth kinetics of M. tuberculosisH37Rv ΔRD1 in BALB/c mice. Mice were infected with 2×10⁶ CFU throughtail injection. Time to death was noted and at day 1, week 4, 8, 14, and22 post-infection, mice were sacrificed to determine the mycobacterialburden in the spleen, liver, and lung. The numbers represent the meansof CFUs in organs derived from three animals. The error bars representthe standard errors of the means. Panel a, Time to death assay in BALB/cmice. Panel b, Spleen. Panel c, Liver. Panel d, Lung.

FIG. 5 a-5 i are micrographs from pathological studies of infectedBALB/c mice. Panels a-c, Lungs from mice infected with 2×10⁶ CFU of M.tuberculosis H37Rv examined at 4, 8 and 14 weeks post-infection. Themild to moderate pneumonia at 4 and 8 weeks (a and b) progressed tosevere consolidating granulomatous pneumonia at 14 weeks post infection(c). Panels d-f, Lungs from mice infected with 2×10⁶ CFU of M.tuberculosis H37Rv ARD1 examined at 4, 8 and 22 weeks post-infectionshowing moderate pneumonia at 8 weeks post-infection (e) and persistentbronchitis and multifocal pneumonitis at 22 weeks post-infection (f).Panels (g)-(i), Mild lung lesions from mice infected with 2×10⁶ CFU ofBCG at 4, 8 and 22 weeks post-infection. Mild focal granulomas scatteredwidely in the lung at each time point with predominately lymphocyticaccumulations in foci at 22 weeks post-infection.

FIG. 6 shows graphs summarizing experiments establishing thatpantothenate auxotrophy leads to attenuation of M. tuberculosis ΔpanCDmutant in SCID mice. Panel A, Survival of BALB/c SCID mice (n=12 pergroup) infected intravenously with 490 CFU of H37Rv (∘) or 210 CFU ofpanCD complementing strain (panCD in single copy integrated into thechromosome)(●) or 3.4×10⁵ CFU of ΔpanCD mutant (▴) or 3.3×10⁵ CFU ofBCG-P (□). Panel B, Bacterial numbers in the spleen (●) and lungs (▴) ofSCID mice infected intravenously with 490 CFU of H37Rv or numbers inspleen (∘) and lungs (Δ) of mice infected with 3.4×10⁵ CFU of ΔpanCDmutant. The results represent means±standard errors of four to five miceper group.

FIG. 7 shows graphs summarizing experiments demonstrating theattenuation, limited growth and persistence of ΔpanCD mutant inimmunocompetent mice. Panel A, Survival of BALB/c mice (n=12 per group)infected with 4.4×10⁶ CFU of wild-type M. tuberculosis H37Rv (∘),3.2×10⁶ CFU panCD complementing strain (panCD in single copy integratedinto the chromosome)(●) or 2.4×10⁶ CFU panCD mutant (▴). Panels B and C,Bacterial loads in spleen and lungs of BALB/c mice infectedintravenously with 4.4×10⁶ CFU wild-type H37Rv (∘) or 3.2×10⁶ CFU panCDcomplementing strain (●) or 2.4×10⁶ CFU ΔpanCD mutant (▴). CFUs wereassayed at various time points on 7H11 agar with or without pantothenatesupplementation where required. The results represent means±standarderrors of four to five mice per group.

FIGS. 8A-8B show graphs summarizing experiments demonstrating theattenuation, limited replication and persistence of ΔnadBC mutant inimmunocompetent mice. Panels A and B, Bacterial loads in lungs andspleen of C57BL/6 mice infected with wild type M. tuberculosis H37Rv (●)or ΔnadBC mutant (∘). Mice were infected intravenously with 10⁶ CFU ofeach strain. CFUs were assayed at various time points on 7H11 agar withor without nicotinamide supplementation where required. The resultsrepresent means±standard errors of four to five mice per group. Panel C,Survival of C57BL/6 mice (n=12 per group) infected with 10⁶ CFU ofwild-type bacteria (●) or 10⁶ CFU of ΔnadBC mutant (∘).

FIG. 9 shows an illustration, map and autoradiograph relating to thepathway for the biosynthesis of pantothenic acid and coenzyme A and itsdisruption in M. tuberculosis. Panel a. The enzymes involved in thebiosynthesis of pantothenic acid and having annotation in the genomicsequence of M. tuberculosis H37Rv are shown in bold numbers: 1) panB,ketopantoate hydroxymethyl transferase; 2) panD,aspartate-1-decarboxylase; 3) panC, pantothenate synthetase; 4) panK,pantothenate kinase; 5) acpS, ACP synthase. Panel b. Map of the panCDgenomic region in the wild type M. tuberculosis H37Rv and the ΔpanCDmutant. Restriction sites and probe location are indicated. Panel c.Southern blot of BssHII-digested genomic DNA from wild-type H37Rv (lane1), two independent clones of ΔpanCD mutant from H37Rv (lanes 2 & 3) andprobed with a 716 bp downstream region flanking the panCD operon.Molecular size marker (in kb) is shown on the left.

FIGS. 10A-10F show graphs summarizing experiments demonstrating thatpantothenate auxotrophy leads to attenuation of ΔpanCD mutant in mice.A. Survival of BALB/c SCID mice (n=12 per group) infected intravenouslywith H37Rv (∘) or panCD-complemented strain (●) or ΔpanCD mutant (A) orM. bovis BCG-P (□). B. Bacterial numbers in the spleen (∘), liver (□)and lung (Δ) of SCID mice infected intravenously with H37Rv or thebacterial numbers in the spleen (●), liver (▪) and lung (▴) of miceinfected with ΔpanCD mutant. C. Survival of immunocompetent BALB/c mice(n=16 per group) infected with H37Rv (∘) or panCD-complemented strain(●) or ΔpanCD mutant (▴). D, E, F. Bacterial numbers in lung (D), spleen(E) and liver (F) of immunocompetent BALB/c mice infected intravenouslywith either H37Rv (∘), panCD-complemented strain (●) or ΔpanCD mutant(▴). Data are means±standard errors of four to five mice per group.

FIGS. 11A-11F show micrographs and graphs summarizing experimentsdemonstrating that the ΔpanCD mutant produces less tissue pathology inlungs of infected BALB/c mice and protects mice against challenge withvirulent M. tuberculosis. A. Severe consolidating granulomatouspneumonia (★) obliterating the normal lung parenchyma at 3 weekspost-infection with H37Rv. B. Severe consolidating granulomatouspneumonia (★) obliterating the normal lung parenchyma at 3 weekspost-infection with the panCD-complemented strain, similar to the wildtype strain. C. Mild lung infection caused by the ΔpanCD mutant at 3weeks post-infection. Localized multifocal granulomas (arrows) scatteredwidely in the lung. Most of the lung is normal alveolar spaces andairways. D. Lung of mouse infected with ΔpanCD mutant examinedhistologically at 23 weeks post-infection. Occasional focal, mildperivascular and interstitial infiltrations composed of predominatelylymphocytes (arrows). Most of the lung is normal alveolar spaces andairways. E, F. The attenuated ΔpanCD mutant protects mice againstaerogenic challenge with virulent M. tuberculosis Erdman. Subcutaneouslyimmunized mice were challenged after 90 days through the aerosol route.The CFU numbers reflect the bacterial burden at 28 days post aerosolchallenge in the lung (E) and spleen (F). Naive mice—black fill; miceinfected with 1 dose panCD—light shade; mice infected with 2 dosespanCD—dark shade; mice infected with BCG-P-unshaded.

FIG. 12 shows autoradiographs of Southern analysis of the NcoI-digestedgenomic DNA isolated from the wild type and the ΔRD1 mutants generatedusing specialized transduction in M. tuberculosis and M. bovis. Lanes:1—M. tuberculosis H37Rv; 2—M. tuberculosis H37Rv ΔRD1; 3—M. tuberculosisErdman; 4—M. tuberculosis Erdman ΔRD1; 5—M. tuberculosis CDC1551; 6—M.tuberculosis CDC1551 ΔRD1; 7—M. bovis Ravenel; and 8—M. bovis RavenelΔRD1. The probed used in the Southern analysis was either DFS (left) orIS6110-specific sequence (right).

FIG. 13 shows graphs summarizing data confirming that deletion of RD1 inM. tuberculosis and M. bovis confers an attenuation of virulence for M.tuberculosis and M. bovis, as indicated by these Time to death curves ofmice infected intravenously with 2×10⁶ CFU mycobacteria. Panel A, SCIDmice infected with M. tuberculosis H37Rv (▪), M. tuberculosis H37Rv ΔRD1(□), M. tuberculosis Erdman (●), M. tuberculosis Erdman ΔRD1 (∘), M.tuberculosis CDC1551 (▴), M. tuberculosis CDC1551 ΔRD1 (Δ), M. bovisRavenel (♦), M. bovis Ravenel ΔRD1 (∇); Panel B, SCID mice infectedintravenously with M. tuberculosis H37Rv (●), M. tuberculosis ΔRD1 (▪),M. tuberculosis ΔRD1::2F9 (▴), M. bovis Ravenel (∘), M. bovis RavenelΔRD1 (□), and M. bovis BCG (Δ); Panel C, BALB/c mice were infected withM. tuberculosis H37Rv (∘), M. tuberculosis ΔRD1 (Δ), and M. bovis BCG(□).

FIGS. 14A-14C are graphs summarizing experiments demonstrating theclearance of the lysine auxotroph in SCID mice. The viable bacterialcounts are shown for the spleens, livers, and lungs of SCID miceinjected intravenously with the lysine auxotroph strain and theprototrophic control strain. Three mice were assayed at each time point.The error bars indicate the standard deviations of the mean values. Notethat the counts at time zero are the counts obtained at 24 hourspost-injection, as described in Example 5. Panels A, B and C show thelog of the viable bacteria in each organ after injection with 1×10⁷ CFUof the Lys⁻ M. tuberculosis mutant mc²3026 (□), or 1×10⁷ CFU of thecomplemented Lys⁺ M. tuberculosis strain mc²3026/pYUB651 (♦).

FIGS. 15A-15C are graphs summarizing experimental results of experimentsthat establish the vaccine efficacy of the M. tuberculosis lysineauxotroph mc²3026. C57Bl/6 mice were injected intravenously with 1×10⁶CFU of the M. tuberculosis lysine auxotroph mc²3026, followed by one ortwo additional injections at 4 week intervals. Five mice were sacrificedweekly after each immunization and the viable bacteria counts of theauxotroph determined in the lungs and spleens. Control mice were given asimilar amount of BCG-Pasteur or only PBST. Shown in Panel A is theclearance of the auxotroph from the lungs of the mice after eachimmunization period; one injection (▪), two injections (⋄), and threeinjections (∘). Three months after the initial immunization thevaccinated and control mice were challenged with virulent M.tuberculosis Erdman by the aerosol route. Five challenge mice weresacrificed following the challenge period and the lung homogenatesplated to check the viable counts of the challenge inoculum. Groups ofvaccinated and control mice were sacrificed at 14, 28, and 42 days laterand the lung and spleen homogenates plated to determine viable colonyforming units. Shown in Panel B are the viable challenge bacteria perlung of mice given one dose of the M. tuberculosis lysine auxotroph, andin panel C, the viable challenge bacteria per lung of mice given twodoses of the auxotroph. Key: Viable challenge bacteria per lung of micegiven the M. tuberculosis lysine auxotroph mc²3026 (▪), BCG-Pasteur (⋄),or PBST (∘). P values are indicated in the figure. Note that the resultsshown here are for the lungs. Similar results (not shown) were obtainedfrom the spleens in all the experiments.

FIG. 16 shows a graph summarizing experiments establishing the survivalcurves of mice immunized three times with the M. tuberculosis lysineauxotroph mc²3026. C57Bl/6 mice were injected intravenously with 1×10⁶CFU of the M. tuberculosis lysine auxotroph mc²3026, followed by twomore injections at 4 week intervals, and challenged as described inExample 5. The percent survival is shown above for mice immunized thricewith the M. tuberculosis lysine auxotroph mc²3026 (▪, 5 mice total),once with BCG-Pasteur (♦, 5 mice), and for the PBST controls (●, 10mice).

FIG. 17 shows graphs summarizing experimental results establishing thatthe virulence of strain mc²6030 is highly attenuated in SCID mice andBALB/c mice.

FIG. 18 shows graphs summarizing experimental results measuring growthof various strains of M. tuberculosis in spleen (Panel A) and lungs(Panel B) of C57BL/6 mice.

FIG. 19 is a graph summarizing experimental results establishing thatimmunization with mc²6020 and mc²6030 protects mice against TB aseffectively as BCG. This graph shows the survival of C57Bl/6 micechallenged with virulent M. tuberculosis Erdman through the aerosolroute three months after a single dose subcutaneous immunization witheither BCG, mc²6020 (ΔlysAΔpanCD) or mc²6030 (ΔRD1ΔpanCD) and comparedto non-immunized naive mice. There were 12 to 15 mice in each survivalgroup.

FIG. 20 is a graph summarizing experimental results establishing thatimmunization with mc²6030 protects CD4 deficient mice from TB. CD4^(−/−)mice were immunized by a single subcutaneous injection of 10⁶ CFU ofeither ΔRD1ΔpanCD (mc²6030) or BCG-P and three months later challengedwith 100-200 CFU of virulent M. tuberculosis through the aerosol routeand compared to non-immunized (naive) CD4^(−/−) controls. Each groupconsists of 10 mice.

FIG. 21 shows graphs summarizing experimental results establishing thatM. tuberculosis double deletion mutants are highly attenuated in SCIDmice. A dose of 10⁵ mc²6020 or mc²6030 were intravenously inoculatedinto SCID mice (10 per group) and time to death assessments wereperformed. While the same dose of M. tuberculosis and BCG killed mice in40 or 90 days, respectively, the mice infected with mc²6020 or mc²6030survived over 400 or 250 days, respectively.

FIGS. 22A-22I are graphs and micrographs of experimental results showingthat mc²6030 is severely attenuated in immunocompromised mice. A.Survival of SCID mice infected intravenously with 10² CFUs of H37Rv (∘)or 10⁵ CFUs of mc²6030 (●). B, C. Bacterial numbers in the spleen (B),lungs (C), and of H37Rv (∘) or mc²6030 (●) infected SCID mice. Theresults represent means±standard errors of four to five mice per group.D. Survival of gamma interferon gene-disrupted (GKO) C57BL/6 mice (n=10mice) infected intravenously with 10⁵ CFUs of H37Rv (0) or mc²6030mutant (●) or M. bovis BCG-P (▴). E. Survival of immunocompetent C57BL/6mice (n=10 mice) infected intravenously with 106 CFUs of H37Rv (∘) ormc²6030 (●). F, G. Bacterial numbers in spleen (E) and lungs (G) ofC57BL/6 mice infected intravenously with H37Rv (∘) or mc²6030 (●). Theresults represent means±standard errors of four to five mice per group.H. Mild perivascular, lymphocytic infiltrates caused by strain mc²6030in C57BL/6 mice at 3 weeks post-infection. I. Severe granulomatouspneumonia in the lungs of C57BL/6 mice infected with H37Rv at 3 weekspost-infection.

FIGS. 23A-23E are graphs of experimental results showing thatvaccination with mc²6030 induces both short-term and long-termprotection in C57BL/6 mice. A, B. Immunocompetent C57BL/6 mice wereimmunized subcutaneously (s.c) with mc²6030 or BCG-P and challenged withvirulent M. tuberculosis Erdman through the aerosol route at 3 monthsafter the initial immunization. The CFU numbers reflect the bacterialburden at 28 days post-aerosol challenge in the lungs and spleen ofinfected mice. C, D. Immunocompetent C57BL/6 mice were immunizedsubcutaneously (s.c) with mc²6030 or BCG-P and challenged with virulentM. tuberculosis Erdman through the aerosol route at 8 months after theinitial immunization. The CFU numbers reflect the bacterial burden at 28days post-aerosol challenge in the lungs and spleen of infected mice.The results represent means±standard errors of five mice per group.**P<0.01, ***P<0.001 indicate statistical differences between theexperimental and unvaccinated control groups. E. Survival ofimmunocompetent C57BL/6 mice (n=10 mice) immunized subcutaneously with asingle dose of mc²6030 (●) or BCG-P (▴) and challenged 3 months laterwith virulent M. tuberculosis Erdman through the aerosol route.Unvaccinated mice served as naive controls (∘).

FIGS. 24A-24F are graphs of experimental results showing thatvaccination with mc²6030 protects and confers greater survival advantageto CD4^(−/−) mice from tuberculous challenge. A, B. Protection inducedby a single dose of mc²6030 in CD4-deficient mice following aerosolchallenge with virulent M. tuberculosis Erdman. The CFU numbers reflectthe bacterial burden at 28 days post-aerosol challenge in the lungs (a)and spleen (b) from 5 mice per group. **P<0.001 indicate statisticaldifferences between the experimental and unvaccinated control groups. C.Survival of CD4^(−/−) mice (n=5 or 6 mice) immunized subcutaneously witha single dose of mc²6030 (●) or BCG-P (▴) and challenged 3 months laterwith virulent M. tuberculosis Erdman through the aerosol route.Unvaccinated mice served as naive controls (∘). D, E. Treatment ofmc²6030-vaccinated CD4^(−/−) mice with anti-CD8 antibody does notabolish the protection seen in mc²6030-vaccinated control antibodytreated CD4^(−/−) mice. The CFU numbers reflect the bacterial burden at28 days post-aerosol challenge in the lungs (D) and spleen (E) from 5mice per group. **P<0.001 indicate statistical differences between theexperimental and unvaccinated control groups. F. Survival of vaccinatedGKO mice following an aerosol challenge with virulent M. tuberculosis.

FIGS. 25A-25F are micrographs of experimental results showing that.mc²6030 vaccinated CD4^(−/−) mice display improved lung pathologyfollowing challenge with virulent M. tuberculosis. A. Severe pneumoniain lung of unvaccinated mice at 28 days post-aerosol challenge, with D,large numbers of M. tuberculosis Erdman organisms demonstrated byacid-fast stain. B. Lung from mouse vaccinate with mc²6030 showingmilder multifocal areas of pneumonia composed of macrophages andnumerous lymphocytes, with E, lower number of M. tuberculosis Erdmanorganisms indicating protection following immunization. C. BCGvaccinated mouse. Similar localized areas of pneumonia adjacent to theairways post-aerosol challenge and F, reduced numbers of acid-fastorganisms similar to mc²6030 vaccinated mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that mycobacteria havingtwo attenuating mutations are safe and protect mammals that aredeficient in CD4⁺ lymphocytes from challenge by virulent mycobacteria.See Examples 6 and 7. This anti-mycobacterial immunity also does notdepend on CD8⁺ cells (Example 7).

Thus, the present invention is directed to methods of treating a mammalthat does not have severe combined immune deficiency but is deficient inCD4⁺ lymphocytes. The methods comprise inoculating the mammal with anattenuated mycobacterium in the Mycobacterium tuberculosis (M.tuberculosis) complex. In these embodiments, the mycobacterium comprisestwo deletions, where a virulent mycobacterium in the M. tuberculosiscomplex having either deletion exhibits attenuated virulence. The mammalin these embodiments can have CD8⁺ lymphocytes that are elevated (as canoccur in AIDS patients), normal or deficient (see Example 7).

The protection afforded by those double mutants in mammals deficient inCD4⁺ lymphocytes is surprising because it was previously believed thatCD4⁺ lymphocytes were crucial in establishing immunity againsttuberculosis (Jones et al., 1993; Scanga et al., 2000). The instantdiscovery establishes that such mutants would be expected to be safe andeffective in providing protection against tuberculosis in individualswith highly compromised immunity, such as individuals with HIV infectionor those taking immunosuppressant drugs (e.g., transplant patients).

As used herein, a mammal that is deficient in CD4⁺ lymphocytes has lessthan about 500 CD4⁺ cells per mm³ of blood. The CD4⁺ deficient mammalcan also have less than about 350 or 200 or 100 or 50 cells per mm³ ofblood. The CD4⁺ deficient mammal can even be devoid of CD4⁺ lymphocytes.A mammal that is deficient in CD8⁺ lymphocytes has less than about 200CD8⁺ cells per mm³ of blood. The CD8⁺ deficient mammal can also haveless than about 100 or 50 or 25 CD8⁺ cells per mm³ of blood. The CD4⁺deficient mammal can also be devoid of CD4⁺ lymphocytes.

The mammal can be inoculated with the mycobacteria in the methods of thepresent invention by any of a number of ways known in the art.Non-limiting examples include oral ingestion, gastric intubation, orbroncho-nasal-ocular spraying. Other methods of administration includeintravenous, intramuscular, intramammary, or, preferably, subcutaneousor intradermal injection. The immunization dosages required can bedetermined without undue experimentation. One or two dosages ofavirulent mycobacteria at 1-2×10⁶ colony forming units (CFU) havepreviously been used, but other dosages are contemplated within thescope of the invention. Multiple dosages can be used as needed toprovide the desired level of protection from challenge.

The above-described mycobacterial compositions can be formulated withoutundue experimentation for administration to a mammal, including humans,as appropriate for the particular application.

Accordingly, the mycobacterial compositions designed for oral, lingual,sublingual, buccal and intrabuccal administration can be made withoutundue experimentation by means well known in the art, for example withan inert diluent or with an edible carrier. The compositions may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral administration, the mycobacterial compositions of the presentinvention may be incorporated with excipients and used in the form oftablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

The mycobacterial compositions of the present invention can easily beadministered parenterally such as for example, by intravenous,intramuscular, intrathecal or subcutaneous injection. Parenteraladministration can be accomplished by incorporating the mycobacterialcompositions of the present invention into a suspension. Suchsuspensions may also include sterile diluents such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents. Parenteral formulationsmay also include antibacterial agents such as for example, benzylalcohol or methyl parabens, antioxidants such as for example, ascorbicacid or sodium bisulfite and chelating agents such as EDTA. Buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose may also be added. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

Rectal administration includes administering the mycobacterialcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the composition in the glycerin, mixing the heated glycerinafter which purified water may be added, and pouring the hot mixtureinto a suppository mold.

The present invention also includes nasally administering to the mammala therapeutically effective amount of the mycobacterial composition. Asused herein, nasally administering or nasal administration includesadministering the composition to the mucous membranes of the nasalpassage or nasal cavity of the patient. As used herein, mycobacterialcompositions for nasal administration of a composition includetherapeutically effective amounts of the composition prepared bywell-known methods to be administered, for example, as a nasal spray,nasal drop, suspension, gel, ointment, cream or powder. Administrationof the composition may also take place using a nasal tampon or nasalsponge.

It is well known in the art that in order to elicit an immune responsewith a live vaccine such as an avirulent mycobacteria, it is preferredthat the vaccine organism can sustain an infection in the immunizedhost, to provide a sustained exposure of the host's immune system to theorganism. Therefore, in various preferred embodiments, the mycobacteriaused in the methods of the invention are capable of sustaining aninfection in the host. The ability to sustain infection can be measuredwithout undue experimentation by any of a number of ways described inthe art. With the mycobacteria used in the methods of the presentinvention, a preferred way of measuring sustained infection is bydetermining whether viable mycobacteria of the inoculated strain willremain resident in an immunocompetent mouse (e.g., BALB/c or C57BL/6strain) for more than four weeks. More preferably, the inoculatedmycobacteria will remain resident in the mouse for at least ten weeks.In the most preferred embodiments, viable mycobacteria of the inoculatedstrain will remain resident in the mouse for at least 20 weeks.

Preferably, the attenuated mycobacteria used in the methods of theinvention are capable of protecting a mammal from challenge by avirulent M. tuberculosis complex mycobacteria. This ability can bedetermined by any of a number of ways provided in the literature. Apreferred method is aerogenically treating an immunocompetent mouse withthe virulent mycobacteria, as described in Examples 1 and 2. Aerogenicchallenge is preferred because that most closely mimics naturalinfection. The skilled artisan would understand that the ability of anavirulent mycobacterium to protect a mouse from challenge from avirulent mycobacterium is indicative of the ability of the avirulentmycobacterium to protect a human, including a human child, fromtuberculosis infection. A more stringent test of an avirulentmycobacterium to prevent infection by a virulent challenge is to use animmunocompromised mammal, e.g. a SCID mouse or a mouse deficient in CD4or interferon γ production.

The scope of the present invention includes the use of mycobacteria inthe M. tuberculosis complex that comprise two attenuating deletions,where at least one of the attenuating deletion was made using geneticengineering. As discussed above, examples of such deletions includedeletions of an RD1 region, deletions of a region controlling productionof a vitamin, and deletions of a region controlling production of anamino acid. These mycobacteria include any in the M. tuberculosiscomplex, including M. africanum, M. bovis including the BCG strain andthe subspecies caprae, M. canettii, M. microti, M. tuberculosis and anyother mycobacteria within the M. tuberculosis complex, now known orlater discovered. Preferred species are M. bovis, including the BCGstrain, and M. tuberculosis, since those species are the most importantas causes of mammalian diseases, such as tuberculosis in humans and M.bovis infection in cows.

In some aspects of these embodiments, at least one of the two deletionsis of the RD1 region (see Example 1). Strains with these deletions canbe determined by any means in the art, preferably by molecular geneticmeans, for example by hybridization methods (e.g., Southern blot using aprobe from the RD1 region) or by amplification methods (e.g., PCR usingprimers to amplify a portion of the RD1 region). An example of an M.tuberculosis RD1 region (from H37Rv) is provided herein as SEQ ID NO:1.The skilled artisan could identify analogous RD1 regions from othermycobacteria in the M. tuberculosis complex without undueexperimentation. Those RD1 regions would be expected to have stronghomology to SEQ ID NO:1, at least 80% homologous to SEQ ID NO:1.However, it is to be understood that virulent mycobacteria in the M.tuberculosis complex can be rendered avirulent by deletions in a portionof the RD1 region. Therefore, use of non-naturally occurringmycobacteria in the M. tuberculosis complex that have a partial deletionin the RD1 region are envisioned as within the scope of the invention,provided the deletion can cause a virulent M. tuberculosis to becomeavirulent. It is expected that such M. tuberculosis with partial RD1deletions can still sustain an infection in a mammal and protect againstchallenge by a virulent M. tuberculosis.

In embodiments where at least one of the deletions is in a regioncontrolling production of a vitamin, the deletion can be in any geneticelement leading to loss of production of the vitamin, includingstructural genes for enzymes involved in the biosynthesis of thevitamin, and genetic control elements such as promoters, enhancers, etc.

Deletion of a region controlling production of any essential vitamin ortheir precursors is contemplated as within the scope of the invention.As used herein, an essential vitamin is defined by its normal usage,that is, a small molecular weight compound that is required as acofactor for the efficient function of an essential enzyme or enzymes.Nonlimiting examples include vitamin A, thiamin (B1), riboflavin (B2),nicotinic acid (niacin)/nicotinamide/nicotinamide adenine dinucleotide(NAD)/nicotinamide adenine dinucleotide phosphate (NADP/coenzyme II),pantothenate (pantothenic acid/B5), pyridoxine (B6), folic acid, B12,biotin, C, D, E and K. Preferred vitamin targets for deletion includenicotinamide and pantothenate (see Example 2). Methods for determiningwhether a mycobacterium has deletions leading to the loss of productionof any of these vitamins are within the scope of the art.

Deletions leading to the loss of any of these vitamins would be expectedto lead to attenuated virulence of an otherwise virulent mycobacteriumin the M. tuberculosis complex. Any of those strains would also beexpected to sustain an infection in a mammal.

Preferred vitamin targets are pantothenate and nicotinamide adeninedinucleotide (NAD)(see Example 2). A preferred pantothenate deletion isof structural genes in the pantothenate biosynthetic operon, mostpreferably the pan C and D genes, the combined mutation being ΔpanCD. Anexample of a deletion of those genes is the deletion of the sequencefrom M. tuberculosis H37Rv provided herein as SEQ ID NO:2. Similarly, apreferred NAD deletion is in the structural genes of the NADbiosynthetic operon, most preferably the nadB and nadC genes, thecombined mutation being ΔnadBC. An example of a deletion in those genesis the deletion of the sequence from M. tuberculosis H37Rv providedherein as SEQ ID NO:3.

In other embodiments, at least one of the attenuating deletions is of aregion controlling production of an amino acid. Preferred examplesinclude deletions of a region controlling production of proline,tryptophan, leucine or lysine. When the amino acid is lysine, apreferred deletion is a ΔlysA deletion, e.g., SEQ ID NO:4.

The mycobacterium deletions can be made by serial in vitro passage of avirulent M. tuberculosis (as the well-known M. bovis BCG was made) andselection for the desired deletion. More preferably, however, thedeletion is made by genetic engineering, since such genetic methodsallow precise control of the deletion being made.

Various methods of making deletions in mycobacteria are known in theart. Nonlimiting examples include specialized transduction (see, e.g.,U.S. Pat. No. 6,271,034, Example 1 and Example 2), and sequentialtwo-step recombination (see Example 1). The latter method can usefullyemploy a sacB selective marker (Example 1).

Since, in preferred embodiments of the invention, the mycobacteriaexhibit attenuated virulence and can sustain an infection in a mammal,these mycobacteria can usefully further employ a foreign DNA stablyintegrated into the genome of the mycobacteria, such that themycobacteria can express a gene product coded by the foreign DNA. See,e.g., U.S. Pat. No. 5,504,005.

Thus, the present invention has wide applicability to the development ofeffective recombinant vaccines in mammals deficient in CD4⁺ lymphocytesagainst bacterial, fungal, parasite or viral disease agents in whichlocal immunity is important and might be a first line of defense.Non-limiting examples are recombinant vaccines for the control ofbubonic plague caused by Yersinia pestis, of gonorrhea caused byNeisseria gonorrhoea, of syphilis caused by Treponema pallidum, and ofvenereal diseases or eye infections caused by Chlamydia trachomatis.Species of Streptococcus from both group A and group B, such as thosespecies that cause sore throat or heart disease, Neisseria meningitidis,Mycoplasma pneumoniae, Haemophilus influenzae, Bordetella pertussis,Mycobacterium leprae, Streptococcus pneumoniae, Brucella abortus, Vibriocholerae, Shigella spp., Legionella pneumophila, Borrelia burgdorferi,Rickettsia spp., Pseudomonas aeruginosa, and pathogenic E. coli such asETEC, EPEC, UTEC, EHEC, and EIEC strains are additional examples ofmicrobes within the scope of this invention from which foreign genescould be obtained for insertion into mycobacteria of the invention.Recombinant anti-viral vaccines, such as those produced againstinfluenza viruses, are also encompassed by this invention. Recombinantanti-viral vaccines can also be produced against viruses, including RNAviruses such as Picornaviridae, Caliciviridae, Togaviridae,Flaviviridae, Coronaviridae, Rhabdoviridae, Filoviridae,Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae,Reoviridae or Retroviridae; or DNA viruses such as Hepadnaviridae,Paroviridae, Papovaviridae, Adenoviridae, Herpesviridae or Poxyiridae.

The use of these methods for administering recombinant vaccines toprotect against infection by pathogenic fungi, protozoa or parasites arealso contemplated by this invention.

The avirulent microbes used in the methods of the present invention arealso contemplated for use to deliver and produce foreign genes thatencode pharmacologically active products that might stimulate orsuppress various physiological functions (i.e., growth rate,immune-stimulating cytokines, blood pressure, etc.) in CD4⁺-deficientmammals. In such microbes, the recombinant gene encodes saidpharmacologically active products.

By immunogenic agent is meant an agent used to stimulate the immunesystem of an individual, so that one or more functions of the immunesystem are increased and directed towards the immunogenic agent.Immunogenic agents include vaccines.

An antigen or immunogen is intended to mean a molecule containing one ormore epitopes that can stimulate a host immune system to make asecretory, humoral and/or cellular immune response specific to thatantigen.

In preferred embodiments, the foreign DNA encodes an antigen, an enzyme,a lymphokine, an immunopotentiator, or a reporter molecule. Preferredexamples include antigens from Mycobacterium leprae, Mycobacteriumtuberculosis, malaria sporozoites, malaria merozoites, diphtheriatoxoid, tetanus toxoids, Leishmania spp., Salmonella spp., Mycobacteriumafricanum, Mycobacterium intracellulare, Mycobacterium avium, Treponemaspp., Pertussis, Herpes virus, Measles virus, Mumps virus, Shigellaspp., Neisseria spp., Borrelia spp., rabies, polio virus, humanimmunodeficiency virus, snake venom, insect venom, and Vibrio cholera;steroid enzymes; interleukins 1 through 7; tumor necrosis factor α andβ; interferon α, β, and γ; and reporter molecules luciferase,β-galactosidase, β-glucuronidase and catechol dehydrogenase.

It has also been discovered that the attenuated mycobacteria discussedabove can safely be used to inoculate mammals deficient in CD8⁺lymphocytes, for example mammals that are immunosuppressed, or mammalsthat have a mutation in a CD8 gene (see, e.g., de la Calle-Marin et al.,2001). See Example 7.

Thus, the invention is also directed to methods of treating a mammalthat does not have severe combined immune deficiency but is deficient inCD8⁺ lymphocytes. The methods comprise inoculating the mammal with anattenuated mycobacterium in the Mycobacterium tuberculosis (M.tuberculosis) complex. In these embodiments, the mycobacterium comprisestwo deletions, where a virulent mycobacterium in the M. tuberculosiscomplex having either deletion exhibits attenuated virulence. Themammals in these methods can have normal levels of CD4⁺ lymphocytes, orhave deficient or elevated levels of CD4⁺ lymphocytes.

These embodiments are entirely analogous to the embodiments discussedabove relating to the treatment of mammals deficient in CD4⁺lymphocytes. This includes the use of any of the above-describedattenuated M. tuberculosis strains and any of the inoculation methodsdescribed above. The methods can also be used in any mammal, includinghumans, such as adults or children.

Additionally, the invention is directed to the use of an attenuatedmycobacterium in the Mycobacterium tuberculosis (M. tuberculosis)complex for the manufacture of a medicament for treatment of a mammalthat does not have severe combined immune deficiency but is deficient inCD4⁺ lymphocytes, or CD8⁺ lymphocytes. In these embodiments, themycobacterium is as described for the embodiments discussed above, i.e.,it comprises two deletions, where a virulent mycobacterium in the M.tuberculosis complex having either deletion exhibits attenuatedvirulence. Methods of manufacture of such medicaments, includingformulations as vaccines, are well known in the art and would notrequire undue experimentation. These uses are suitable for any mammal,including humans, such as adults or children.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols inMolecular Biology” Volumes I-IV Ausubel, R. M., ed. (1997); and “CellBiology: A Laboratory Handbook” Volumes I-III J. E. Celis, ed. (1994).

EXAMPLE 1 Mycobacterium Tuberculosis Having an RD1 Deletion hasAttenuated Virulence and Protects Against Tuberculosis

This example describes experimental methods and results that establishthat deleting the RD1 region from a virulent M. tuberculosis attenuatesthe virulence of the M. tuberculosis in both immunocompetent andimmunocompromised mice, and protects against subsequent challenge by avirulent M. tuberculosis.

Materials and Methods

Media and Cultures. The mycobacterial strains M. tuberculosis H37Rv, M.tuberculosis Erdman and M. bovis BCG Pasteur were obtained from theTrudeau Culture Collection (Saranac Lake, N.Y.). They were cultured inMiddlebrook 7H9 broth and 7H10 agar supplemented with 10% OADC, 0.5%glycerol, and 0.05% Tween 80. Cyclohexamide, which does not affectmycobacterial growth, was added to the 7H10 agar medium at 0.1% to avoidfungal contamination. To examine the colony morphology of mycobacteria,Tween 80 was not added to 7H10 agar medium. The acriflavin resistantstrain (Hepper and Collins, 1984) of M. tuberculosis Erdman grew in thepresence of 20 μg of acriflavin per ml of medium.

DNA manipulation and construction of M. tuberculosis ΔRD1. The followingfour primers were used to amplify upstream and downstream flankingsequences (UFS and DFS, respectively) for the construction of the RD1deletion mutants. UFS was amplified using TH201: GGGGGCGCACCTCAAACC (SEQID NO:5) and TH203: ATGTGCCAATCGTCGACCAGAA (SEQ ID NO:6). DFS wasamplified using TH203: CACCCAGCCGCCCGGAT (SEQ ID NO:7), and TH204:TTCCTGATGCCGCCGTCTGA (SEQ ID NO:8). Recognition sequences for differentrestriction enzymes were included at the ends of each primer to enableeasier manipulation.

The unmarked deletion mutant of M. tuberculosis H37Rv, mc²4004, wasgenerated by transformation (Snapper et al., 1988) using a sacBcounterselection (Pelocic et al., 1996; Pavelka and Jacobs, 1999).Specifically, the plasmid pJH508 was created by first cloning UFS intoKpnI and XbaI sites, then cloning DFS into EcoRI and HindIII sites ofpJH12, a pMV261-derived E coli-Mycobacteria shuttle plasmid, to createpJH506 in which UFS and DFS flanked a green fluorescent protein gene(GFPuv, Clonetech) whose expression was driven by the M. leprae promoter18 Kd. The UFS-gfp-DFS cassette was sub-cloned into the EcoRV site ofplasmid pYUB657 to create pJH508. The first homologous recombinationinvolved the identification of hygromycin resistant colonies, resultingfrom the transformation of M. tuberculosis with pJH508. Southernanalysis of the NcoI digested DNA isolated from hygromycin resistantcolonies probed with UFS or DFS, confirmed the presence of a single copyof pJH508 inserted into the M. tuberculosis genome. The transformantidentified was then grown in 7H9 broth to saturation, to allow thesecond homologous recombination to occur, resulting in recombinants thatcould be selected by plating the culture on 7H10 plates, supplementedwith 3% sucrose. Both Southern analysis and PCR of the DNA isolated fromsucrose resistant colonies confirmed the RD1 deletion.

Specialized transduction (Bardarov and Jacobs, 1999), amycobacteriophage-based method for the delivery of homologous DNAconstructs using conditionally replicating shuttle phasmids (Jacobs etal, 1987; Bardarov and Jacobs, 1999; Carriere et al., 1997), has beenused successfully for M. tuberculosis (Glickman et al., 2000; Glickmanet al., 2001; Raman et al., 2001). Specifically, a transducing phagephAEKO1 was constructed by inserting UFS and DFS into pJSC347, flankinga hygromycin cassette, to create pJH313. pJH313 was digested with PacIand ligated to phAE159, a temperature sensitive mycobacteriophagederived from TM4. The transduction was performed by growing M.tuberculosis to an O.D.₆₀₀ of 0.8, washing twice with MP buffer,re-suspending into an equal volume of MP buffer and mixing with thetransducing phage phAEKO1 at an MOI of 10. The mixtures were incubatedat 37° C. overnight, then plated on 7H10 plates supplemented withhygromycin at 50 μg/ml. Hygromycin resistant colonies were analyzed byPCR and Southern hybridization, as described above, to confirm thedeletion of RD1.

Complemetation analyses was performed using the integration proficientcosmids (Pascopella et al., 1994; Lee et al., 1991) pYUB412 made by S.Bardarov, a library made by F. Bange, and cosmid identified andgenerously provided by S.T. Cole.

Results

Genetic engineering of M. tuberculosis mutants with RD1 deletions. TheRD1 (region of difference) region has been defined as the specific 9454bp of DNA that is present in virulent M. tuberculosis and M. bovis, butabsent in BCG (Mahairas et al., 1996). The annotation of RD1 predictsthat the deletion would disrupt 9 genes encoding ORF's (Id.; Cole etal., 1998). Five of the 9 ORF's have no known functions (Rv3871, Rv3876,Rv3877, Rv3878 and Rv3879c), two genes encode members of the PE/PPEfamily (Rv3872/Rv3873), and two genes encode the secreted proteins Cfp10(Berhet et al., 1998) and Esat6 (Andersen et al., 1991)(Rv3875) (FIG.1). To test if the RD1 region is essential for virulence in M.tuberculosis, it was necessary to 1) delete the RD1 region from virulentM. tuberculosis strains, 2) demonstrate loss of virulence and 3) restorevirulence by complementation with the RD1 DNA. The RD1 deletion (ΔRD1)was successfully introduced into M. tuberculosis by two differenttechniques, utilizing both a plasmid that allows two-step sequentialrecombination to make an unmarked deletion, and specialized transduction(FIG. 1 a-c). For both methods, the same 1200 bp on each side of the RD1deletion were cloned into the appropriate plasmid or phage vector andthen introduced into M. tuberculosis H37Rv by transformation or phageinfection. An unmarked RD1 deletion mutant of M. tuberculosis H37Rv,mc²4004, was constructed, purified, and has the advantage thatadditional mutations can be readily added to it. In addition, the RD1deletion was successfully engineered in the H37Rv and Erdman strains ofM. tuberculosis using a specialized transducing phage. Since TM4 phageshave been shown to infect over 500 clinical M. tuberculosis isolates(Jacobs et al., 1987), it should be possible to introduce the RD1deletion into any M. tuberculosis isolate of interest.

M. tuberculosis H37Rv ΔRD1 is attenuated for virulence. To test if theRD1 deletion causes an attenuating phenotype in M. tuberculosis, the M.tuberculosis H37Rv ΔRD1 (mc²4004) was introduced into immunocompromisedmice possessing the SCID (severe combined immunodeficiency) mutation.Groups of ten mice were injected intravenously with either 2×10⁶ M.tuberculosis H37Rv or M. tuberculosis H37Rv ΔRD1 and three mice pergroup were sacrificed 24 hours later to verify the inoculation doses.All of the SCID mice infected with the parental M. tuberculosis H37Rvstrain died within 14 to 17 days post infection (FIG. 2 a). In contrast,the SCID mice infected with the same dose of M. tuberculosis H37Rv ΔRD1were all alive at 35 days post-infection demonstrating a markedattenuation of the strain. To prove that the attenuation was due to theRD1 deletion, mc²4004 was transformed with an integrating plasmidcontaining the RD1 region from M. tuberculosis H37Rv. SCID mice injectedintravenously with 2×10⁶ of the transformed strain died 13 to 16 dayspost-infection (FIG. 2 a), thereby, establishing that the genes in theRD1 deletion complemented the attenuating phenotype.

To further characterize the attenuating phenotype of the RD1 deletion inmc²4004, we compared the virulence of M. tuberculosis H37Rv andBCG-Pasteur to M. tuberculosis H37Rv ΔRD1 with time-to-death experimentsin SCID mice following injections with 10-fold varying inocula. Groupsof 10 mice were injected intravenously, each mouse receiving from 2×10³to 2×10⁶ CFU. FIG. 2 b shows that the SCID mice succumbed to theinfection with all three mycobacterial strains. However, the SCID micesuccumbed to an M. tuberculosis H37Rv intravenous infection within 2 to5 weeks, in a dose dependent manner. In the same time frame, the SCIDmice did not succumb to infection with M. tuberculosis H37Rv ΔRD1 untilweek 7, and only then, with the high dose of 2×10⁶ CFU. Mice receiving2×10³ CFU M. tuberculosis H37Rv ΔRD1 survived longer than 14 weeks postinfection, the survival rate of which coincided with the mice receiving2×10⁶ CFU of M. bovis BCG. Thus, these experiments established that M.tuberculosis H37Rv ΔRD1 was significantly more attenuated than itsparent, but not as attenuated as BCG-Pasteur in the immunocompromisedmice.

Colonial morphotypes of M. tuberculosis H37Rv ΔRD1. The M. tuberculosisH37Rv ΔRD1 mutant was generated independently three times from thesingle crossover construct (mc²4000) and upon subculturing, consistentlyyielded a 20 to 50% mixture of two colonial morphotypes on Middlebrookmedium without Tween 80 (FIG. 3 a). One morphotype was a smooth (S)phenotype that was flat and corded (like the parental M. tuberculosisH37Rv strain) and the second was a rough and raised (R) phenotype.Repeated subculturing of either the R or S colonies continued to yieldboth colonial morphotypes, but with a distribution of approximately 80%smooth and 20% rough colonies. The distinction of these two types ofmorphology could be noted even when the colonies were less than twoweeks old as the rough colonies were constricted and elevated with onlya small portion of the base of the colony attached to the agar, whilethe smooth colonies tends to be flattened and spread out. When coloniesgrew older, e.g. 6 weeks old, the rough colonies remained moreconstricted compared to those of smooth colonies. The rough coloniesexhibited large folds on the surface (FIG. 3 f, g), as compared to thoseof the smooth colonies that exhibited small wrinkles (FIG. 3 e).

Interestingly, in 1929, Petroff et al. reported a similar property foran early-derived BCG strain (Petroff et al., 1929) and proposed that theattenuation phenotype of BCG was not stable. Calmette disputed that theavirulent phenotype reverted and postulated that Petroff et al. hadacquired a contaminating virulent strain. Southern analysis of R and Scolonies revealed each morphotype has the same RD1-deleted genotype(FIG. 3 d). Furthermore, complementation of M. tuberculosis H37Rv ΔRD1with the RD1 region restored the mutant phenotype back to the homogenousparental S phenotype (FIG. 3 a-c). These results suggest that thevariable morphotypes resulted directly from the RD1 deletion thusdissociating a direct correlation of virulence with morphotype.

The M. tuberculosis H37Rv ΔRD1 is highly attenuated in immunocompetentBALB/c mice. To further assess the pathogenicity, survival, growthkinetics, and the histopathological analysis of the M. tuberculosisH37Rv ΔRD1 mutant, we compared the parental M. tuberculosis H37Rv toBCG-Pasteur strains in BALB/c mice. In survival studies, greater than50% BALB/c mice had died at 14 weeks post i.v. infection with 2×10⁶ CFUsof M. tuberculosis H37Rv strain (FIG. 4 a). In contrast, all miceinfected with a similar dose of either BCG or M. tuberculosis H37Rv ΔRD1survived for longer than 22 weeks. These results were substantiated in aseparate experiment in which a group of 11 BALB/c mice were infectedwith 1×10⁵ CFU of M. tuberculosis H37RY ΔRD1 and 9 of 11 mice (81%)survived greater than 9 months post-infection (data not shown). WhileBCG and M. tuberculosis H37Rv ΔRD1 showed similar survival results, thegrowth relative kinetics in mouse organs differed substantially. BCGgrew in a limited fashion in lungs, liver and spleen in BALB/c mice andwas cleared to undetectable levels by week 12 (FIG. 4 b-d). In contrast,the M. tuberculosis H37Rv ΔRD1 strain grew in a fashionindistinguishable from the parental M. tuberculosis H37Rv in all mouseorgans for the first 8 weeks. Thereafter, mice infected with theparental M. tuberculosis failed to contain the infection leading tomortality. Strikingly, mice infected with the M. tuberculosis H37Rv ΔRD1showed a definite control over infection resulting in significantlyprolonged survival of mice (FIG. 4 b-d).

The differing survival data of the three strains was clearlysubstantiated by histopathological analysis. M. tuberculosis H37Rv ΔRD1caused less severe organ damage in the lung, liver and spleen than thehighly virulent parent strain M. tuberculosis H37Rv. M. bovis BCG wasthe least virulent of the three strains. Based on histopathologicalevaluation, the mortality in mice infected with the wild type M.tuberculosis H37Rv (documented above and in FIG. 4 a) was caused byworsening pneumonia, hepatitis and splenitis (FIG. 5 a-c). Mice examinedat 14 weeks post-infection had developed severe lobar granulomatouspneumonia. Acid fast staining demonstrated large numbers of M.tuberculosis H37Rv, often in clumps, throughout the lung. The livers andspleens showed a severe diffuse granulomatous inflammation.

Histopathological examination further demonstrated that M. tuberculosisH37Rv ΔRD1 was attenuated in virulence compared to the parent strain M.tuberculosis H37Rv (FIG. 5 d-f). In contrast to the rapidly progressiveinfection with the parent strain M. tuberculosis H37Rv, the lung lesionscaused by M. tuberculosis H37Rv ΔRD1 were maximal in mice examined at 8weeks post-infection. Consolidating granulomatous pneumonia involved anestimated 25-30% of the lung in these mice. Numerous organisms weredemonstrated by acid fast staining. The pneumonia subsequently underwentpartial resolution. By 14 weeks, and again at 22 weeks post-infection,the lungs showed peribronchial and perivascular inflammatory cellaccumulations and focal, generally non-confluent, granulomas now with aprominent lymphocytic infiltration. The numbers of acid fast organismswere reduced. Liver lesions consisted of low numbers of scatteredgranulomas. Spleens were smaller, with persistent granulomas in the redpulp.

Mice infected with M. bovis BCG showed mild lesions in the lung, liverand spleen at all time points (FIG. 5 g-i). At longer time intervalspost-infection the lesions were fewer in number, smaller with prominentlymphocytic infiltrations. At 14 weeks post-infection, M. bovis BCG wasbelow the level of detection by acid fast staining. In summary, whereasM. tuberculosis H37Rv ΔRD1 initially grew in a manner similar to theparental M. tuberculosis H37Rv, this RD1 mutant was limited in theextent of spread of infection, particularly in the lung. This contrastedto the extensive and severe damage caused by the parent strain. Thesubsequent resolving granulomas, localization of the lesions and changesin the composition of the inflammatory cell infiltrations suggested thatthe mice mounted an effective immune response to combat M. tuberculosisH37Rv ΔRD1 infection and thereby reduced the numbers of viableorganisms.

M. tuberculosis H37Rv ΔRD1 protects mice against aerosolized M.tuberculosis challenge. To test the potential of M. tuberculosis H37RvΔRD1 to immunize mice and protect against tuberculous challenge, we usedthe model of subcutaneous immunization followed by aerosol challengewith virulent M. tuberculosis. Our initial studies in C57BL/6 micemonitored the growth the M. tuberculosis H37Rv ΔRD1 strain over an84-day period. Groups of mice (5 mice per group) were vaccinatedsubcutaneously (sc) either once or twice, 6 weeks apart, with 10⁶ CFU M.tuberculosis H37Rv ΔRD1 organisms. Additional mice were infectedintravenously (iv) with the same dose of the RD1-deleted strain in orderto examine the pathogenicity in C57BL/6 mice.

As seen in Table 1, M. tuberculosis H37Rv ΔRD1 persisted in the lungs,liver, and spleen for 3 months at moderate levels of infection but theorganisms failed to grow substantially in the lungs and spleens of micethat had been inoculated iv. In contrast, reduced persistence anddecreased concentrations of M. tuberculosis H37Rv ΔRD1 organisms weredetected in organ homogenates prepared from mice that had beenvaccinated sc. For the groups of mice that had been immunized with onlyone dose sc., low levels of M. tuberculosis H37Rv ΔRD1 bacilli wererecovered from the spleen after 28 and 56 days post-vaccination;however, no splenic mycobacteria were detected 84 days after the sc.injection. Importantly, the concentration of M. tuberculosis H37Rv ΔRD1organisms in the lungs after the sc. immunizations was below thethreshold of detection (<100 CFUs per organ) for the CFU assay at nearlyall time points during the three month study.

TABLE 1 Growth kinetics in C57BL/6 mice. Lung (Log CFU) Spleen (Log CFU)Weeks i.v. s.c. s.c. (2×) i.v. s.c. s.c. (2×) 4 5.86 ± 0.10 <2 not done5.73 ± 0.05 2.41 ± 0.26 not done 8 5.79 ± 0.07 <2 2.52 ± 0.34 5.37 ±0.04 3.12 ± 0.40 3.62 ± 0.29 12 5.61 ± 0.09 <2 <2 5.40 ± 0.05 <2 3.52 ±0.22 Mice were infected with 10⁶ M. tuberculosis H73Rv ΔRD1 by differentroutes. The data are presented as mean ± standard error of the mean.

Three months after the sc. vaccinations with the ΔRD1 strain, groups ofmice were challenged aerogenically with a low dose (50 CFUs) of anacriflavin-resistant strain of M. tuberculosis Erdman. The use of adrug-resistant challenge strain permitted the differentiation of thechallenge organisms from the sensitive vaccine population. As controls,other groups of mice were immunized Sc. with 10⁶ CFUs of BCG Pasteur.The protective responses induced by the M. tuberculosis H37Rv ΔRD1vaccination were evaluated by assessing the relative growth of theacriflavin-resistant challenge organisms in naïve, BCG vaccinated, andM. tuberculosis H37Rv ΔRD1 immunized mice and by comparing the relativepost-challenge lung pathology in the experimental groups and the naivecontrols. As seen in Table 2, the growth of the drug-resistant challengeorganisms was substantially lower in the lungs of animals vaccinatedwith BCG or the M. tuberculosis H37Rv ΔRD1 vaccine. Significantreductions in the lung CFU values in the vaccinated animals (relative tonaive controls) could be detected both 28 and 56 days after thechallenge. Dissemination to the spleen was also significantly limited inall of the vaccination groups with the most substantial differences(−1.4 log₁₀ CFUs compared to the naives) being detected during the firstmonth post-challenge. While significant differences in the growth of themycobacterial challenge was identified between unvaccinated andvaccinated mice, the rate of proliferation of the acriflavin-resistantchallenge strain in all the experimental groups (BCG sc or M.tuberculosis H37Rv ΔRD1 1 or 2 doses sc) was nearly identical and notstatistically different.

TABLE 2 M. tuberculosis ΔRD1 and BCG protect C57BL/6 mice from areosolchallenge with M. tuberculosis Erdman Lung (Log CFU) Spleen (Log CFU)Day 28 Day 56 Day 28 Day 56 Naive 4.77 ± 0.06 4.11 ± 0.05 3.57 ± 0.213.20 ± 0.16 BCG (1×) 3.96 ± 0.20 3.80 ± 0.08 2.18 ± 0.18 2.48 ± 0.23ARD1 (1×) 3.97 ± 0.39 3.71 ± 0.06 2.12 ± 0.12 2.60 ± 0.25 ARD1 (2×) 3.96± 0.15 3.66 ± 0.09 2.21 ± 0.15 2.22 ± 0.16 Immunizations were performedsubcutaneously once (1×) or twice (2×) with 2 × 10⁶ CFUs of thevaccinating strains. Three months later, vaccinated animals wereaerogenically challenged with 50 CFUs/mouse of acriflavin resistant M.tuberculosis Erdman. The growth of the bacterial challenge was monitored28 and 56 days post infection by plating on Middlebrook 7H11 platescontaining 20 μg/ml acriflavin and using procedures previously described(Delogu et al., 2002).Discussion

The M. tuberculosis H37Rv ΔRD1 mutant strain shares significantproperties with BCG including: 1) a significant attenuation of virulencein mice, 2) the ability to generate variable colonial morphotypes, and3) the ability to protect mice against aerogenic tuberculosis challenge.These properties, and the observation that RD1 is the only deletioncommon to all BCG substrains, makes it likely that the RD1 deletion isthe primary attenuating mutation. It remains to be determined if asingle gene or a number of genes in this region causes the attenuatedphenotype. The variable colonial morphotype switch does suggest that aprotein regulating cell wall biogenesis is affected. Notably, definedmutations affecting the cyclopropanation of mycolic acids (Glickman etal., 2000) or the synthesis or export of phthiocerol dimycoseroate (Coxet al., 1999) have been found to correlate with decreased virulence andaltered colony morphotypes in M. tuberculosis and thus representattractive candidate genes that might be regulated by an RD1-encodedgene. The M. tuberculosis ΔRD1 mutant provides a precisely definedbackground strain by which to determine virulence and colony morphologyrelated genes.

BCG is currently the only antituberculous vaccine available for use inhumans. In many animal models, BCG has been shown to induce protectiveimmunity against M. tuberculosis challenge (Opie and Freund, 1937;Hubbard et al., 1992; Baldwin et al., 1998) and in addition, hasdemonstrated protection against the most severe and fatal form of TB inchildren (Rodrigues et al., 1991). However, BCG has shown variableefficacy in protecting adults from pulmonary TB (Tuberculosis PreventionTrial, 1980; Hart and Sutherland, 1977; Bloom and Fine, 1994). Due tothe uncertain efficacy of BCG, particularly in TB-endemic countries, thedevelopment of improved tuberculosis vaccines has become aninternational research priority.

Our challenge studies have demonstrated that the protective immuneresponses elicited by immunization with M. tuberculosis H37Rv ΔRD1 inmice are at least as strong as the protective responses induced byvaccination with BCG. The M. tuberculosis H37Rv ΔRD1 mutant also retainsthe BCG-associated property of limited spread to the lung followingsubcutaneous immunization. Restricted dissemination of the ΔRD1 mutantto the lung suggests it should have a favorable overall safety profile.Also, the unmarked mutant of M. tuberculosis H37Rv ΔRD1 provides asingle deletion strain whereby other attenuating mutations can bereadily engineered. Since the risk of reversion to wild-type virulencedecreases substantially with each additional attenuating mutation, M.tuberculosis mutants harboring deletions in two or three separategenetic loci should provide a much safer vaccine for long term use.

M. tuberculosis mutants with RD1 deletions represent attractivecandidates as novel vaccines for TB prevention. These mutants, derivedfrom a single mutagenic event from the parental M. tuberculosis strain,replicate more efficiently in vivo than BCG, especially early ininfection. This enhanced rate of proliferation for the RD1-deletedstrains, relative to BCG, may lead to the induction of increasedprotective immunity in humans, after vaccination with M. tuberculosisH37Rv ΔRD1. Moreover, they could also be more immunogenic as there existat least 129 ORFs present in M. tuberculosis H37Rv that are absent fromM. bovis (Behr et al., 1999). Since some of these ORFs are likely toencode regulatory proteins affecting the expression of other genes,there could be hundreds of antigens expressed in M.tuberculosis-infected cells that are absent from BCG-infected cells.Thus, RD1 deletion mutants constructed from human tubercle bacilli couldprotect humans against disease substantially better than BCG.

EXAMPLE 2 Vitamin Auxotrophs of Mycobacterium Tuberculosis areAttenuated and Protect Against Tuberculosis

This example describes experimental methods and results that establishthat deleting genes that control vitamin production in a virulent M.tuberculosis causes the M. tuberculosis to become avirulent and sustainan infection in mammals, and protect the mammal against challenge with avirulent M. tuberculosis.

Given the importance of NAD and nicotinamide (vitamin B3) andpantothenate (vitamin B5) as cofactors involved in carbon utilization,energy transduction (Abiko, 1975; Jackowski, 1996) and the biosynthesisof the complex lipid cell wall of M. tuberculosis, we hypothesized thatmutations in the biosynthetic pathways for NAD and pantothenate couldlead to the generation of mutant strains that retain a limited abilityto replicate and subsequently get cleared within the host tissues. In M.tuberculosis, the nadABC operon controls the de novo biosynthesis ofNAD. Similarly, the panC and panD genes that are organized in an operoncontrol the rate-limiting step in the de novo biosynthesis ofpantothenate. We constructed deletion mutants of M. tuberculosis in thenadBC and panCD genes using specialized transduction, as described inExample 1. The mutant strains mc²3122 (ΔnadBC) and mc²6001 (ΔpanCD) wereauxotrophic for nicotinamide and pantothenate respectively. The in vitroreversion frequencies of the respective mutations were found to be lessthan 10⁻¹⁰ events per generation.

The safety and attenuation of ΔnadBC and ΔpanCD auxotrophic mutants wereassessed by infection of immune-compromised SCID mice. SCID miceinfected with wild-type M. tuberculosis and the ΔnadBC mutant succumbedto infection in about 5 weeks (data not shown). This result clearlyindicates that in the absence of T-cell immunity, intermediates of NADbiosynthetic pathway, such as nicotinamide, are readily available in themacrophages to support the growth of the ΔnadBC mutant. In contrast allmice infected with the ΔpanCD mutant survived longer than 30 weeks,demonstrating the severe attenuation of this mutant strain. The fullvirulence phenotype was restored when the panCD wild type alleles wereintegrated into the chromosome of the ΔpanCD mutant in single copy,suggesting the observed attenuation in ΔpanCD to be due to therequirement of pantothenate for growth and not due to polar effects ofthe mutation on downstream genes. SCID mice infected with the same doseof conventional BCG-Pasteur vaccine strain succumbed to infection within80 days (FIG. 6A) in accordance with earlier reports (Guleria, 1996).Enumeration of bacterial burdens in SCID mice infected with wild type M.tuberculosis H37Rv and the complementing strain (panCD in single copyintegrated into the chromosome) showed a rapid increase in bacterialnumbers in spleen, liver and lung before they succumbed to infection. Incontrast, mice infected with ΔpanCD mutant, showed an initial drop inbacterial numbers in spleen and liver followed by a steady increase toreach 10⁸ in the lungs at 160 days, at which time all mice were stillalive (FIG. 6B).

Having demonstrated the significant attenuation of ΔpanCD mutant, wesought to address the in vivo growth characteristics of this mutant inimmune-competent BALB/c mice. All BALB/c mice infected with H37Rvsuccumbed to infection by day 25 with a MST of 22 days. Similarly, miceinfected with the panCD-complemented strain were highly virulent with100% mortality between 3-8 weeks post-infection similar to the wild typestrain, with a MST of 28 days. In contrast, all mice infected withΔpanCD mutant survived for over 33 weeks demonstrating the severeattenuation phenotype of this mutant in immune-competent mice (FIG. 7A).Interestingly, bacterial enumeration at three weeks post infectionshowed 1 log increase in the ΔpanCD numbers in lungs followed by a stateof persistence with the onset of adaptive immune response. This growthcharacteristic was observed only in the lung but not in spleen or liver(FIG. 7B,C). A desirable trait of an effective live attenuated vaccinestrain is its ability to grow within the host in a limited fashion inorder to produce in vivo all the important protective antigens(McKenney, 1999; McKenny, 2000; Kanai, 1955). The ΔpanCD mutant exhibitsthis characteristic in the lung, which is the primary site of infectionin humans and does not get cleared over a prolonged period in all thethree organs. The earlier auxotrophs of M. tuberculosis failed to growin any of the organs and hence failed to adequately protect againstexperimental challenge in guinea pigs (Jackson, 1999), or mice.

The ability of the ΔpanCD mutant to exhibit limited growth in the lunguntil the onset of adaptive immune response suggests that anunidentified putative pantothenate permease is able to transport thisnutrient into resting macrophages, as in the SCID mice. Asodium-dependent pantothenate permease actively transports pantothenateinto the cell of Escherichia coli (Vallari and Rock, 1985; Jackowski andAlix, 1990), Plasmodium falciparum (Saliba and Kirk, 2001) and mammals.Subsequent activation of macrophages leads to restricted supply of thisnutrient within the phagosome resulting in growth arrest of the mutant.Pantothenic acid or its derivatives have been reported to conferresistance to radiation and oxidative stress by virtue of their role inbiosynthesis of CoA and also by indirectly increasing the cellularsupply of glutamate, a precursor of glutathione (Slyshenkov, 1995).Pantothenate kinase (PanK) mutants of Drosophila display membranedefects and improper mitosis and meiosis due to decreased phospholipidbiosynthesis (Afshar et al., 2001). The disruption of de novopantothenate biosynthesis causes an increased susceptibility of theΔpanCD mutant to reactive oxygen and nitrogen intermediates that arereleased within activated macrophages.

Having observed the ΔnadBC mutant to be non-attenuated in SCID mice, wechose to study the in vivo growth kinetics of this mutant in the moreresistant C57BL/6 mice background. During the first three weeks ofinfection, the number of wild type and mutant bacteria recovered fromall three organs showed little or no difference. Their numbers graduallyincreased in the lungs to reach 10⁶. However, with the onset of adaptiveimmune response at three weeks, when the growth of bacteria in the lungsof mice infected with H37Rv became constant and tightly controlled,bacterial load in the lungs of mice infected with ΔnadBC mutant showed aconstant tendency for clearance to reach more than 1.5 log drop in thebacterial numbers compared to mice infected with wild type strain (FIG.8A). This difference was preserved up to 24 weeks following infection.

The reduced ability of the ΔnadBC mutant to sustain an infection wasaccompanied by attenuated virulence clearly seen from the survivalexperiment (FIG. 8C). While all mice infected with the wild type strainsuccumbed to infection between day 90 and 179 (MST 116 days) all miceinfected with the ΔnadBC mutant (n=12) remain alive for a period of morethan 8 months (FIG. 8C).

Our observation of the attenuation phenotype of ΔnadBC mutant becameobvious only after the onset of immune response, suggesting that oncethe macrophages become activated, they restrict the amount of availableNAD or NAD intermediates causing a restricted growth of the mutantstrain. This would be in agreement with the recently reportedobservations that a significant part of antimicrobial function of themacrophages could be attributed to the IFN-γ promoted enhancedexpression of indolamine 2-oxygenase (IDO), the inducible enzymecontrolling L-tryptophan catabolic pathway causing an almost completedepletion of L-tryptophan pool. The enhanced catabolism of L-tryptophanleads to increased de novo biosynthesis of NAD needed to protect thecells from the free radicals formed as a result of macrophageactivation. Recently, several studies have demonstrated the involvementof the tryptophan catabolism in the antimicrobial mechanisms of theactivated macrophages. Induction of IDO was found responsible for theinhibition of intracellular growth of Toxoplasma, Leishimania,Legionella and Chlamydia. The restricted intracellular growth of ΔnadBCmutant could be explained with the very little amount of free NAD or NADintermediates available within the activated macrophages.

Having established the safety and persistence of ΔpanCD and ΔnadBC inimmunocompetent mice, the protective efficacy of these mutants wereevaluated using an aerosol challenge model with virulent M.tuberculosis, using the methods described in Example 1. The aerosolroute of infection was chosen, as this is the natural route of infectionin humans. To assess the capacity of the auxotrophic vaccines torestrict growth of virulent M. tuberculosis in the organs of infectedmice, bacterial numbers were enumerated one month post-infection in lungand spleen. See Table 3. In the unimmunized controls, bacterial numbersrose rapidly in the spleen and lungs, in contrast mice infected with asingle dose of ΔpanCD displayed significant reduction in bacterialnumbers in the spleen and lung (p<0.05, in comparison to unimmunizedcontrols). Mice given two doses of ΔpanCD displayed a statisticallysignificant reduction in the bacterial numbers to 4.5 log units in thelung (p<0.01) and 3.7 log units in the spleen (p<0.05). Mice vaccinatedwith BCG showed comparable reduction in bacterial burden in the lung andspleen to 3.3 log units and 4.7 log units respectively (p<0.01). Miceimmunized with one or two doses of ΔnadBC mutant conferred statisticallysignificant protection (p<0.01 in comparison to unimmunized group) thatis comparable to the protection afforded by BCG vaccination.Interestingly, mice immunized with the ΔnadBC mutant showed nodetectable CFUs in the spleen suggesting that the vaccination completelyprevented the hematogenous spread of wild type M. tuberculosis followingaerosol challenge.

TABLE 3 Table 3. The attenuated M. tuberculosis ΔnadBC and ΔpanCDmutants protect against aerogenic challenge with M. tuberculosis Erdman.Groups of C57BL/6 mice (5 mice per group) were vaccinated subcutaneouslyeither once or twice (6 weeks apart) with 10⁶ CFUs of mutant strains.Control mice were vaccinated subcutaneously with 10⁶ CFUs of BCG-Pasteur. Three months after the initial immunization with either ΔnadBCor ΔpanCD mutant or BCG, the mice were aerogenically challenged withapproximately 100 CFUs of acriflavin-resistant M. tuberculosis Erdman(Ac^(r)MTB) strain as described earlier (Collins, 1985) After 28 days,the challenged mice were sacrificed, and the lungs and spleens ofindividual mice were removed aseptically and homogenized separately in 5ml of Tween 80-saline using a Seward stomacher 80 blender (Tekmar,Cincinnati, OH). The homogenates were diluted serially in Tween 80saline and plated on Middlebrook 7H11 agar with or without appropriatesupplements as required. Samples from the BCG-vaccinated controls wereplated on 7H11 agar containing 2 mg of thiophenecarboxylic acidhydrazide (Sigma Chemical Co., St Louis, MO) per ml to inhibit growth ofany residual BCG. The CFU results were evaluated using the one-way ANOVAanalysis of the GraphPad InStat program. The numbers in paranthesisrepresent the differences between naïve and vaccinated organ CFUs.Experimental Group Lung CFUs (log₁₀) Spleen CFUs (log₁₀) A Naive 4.05 ±0.21 3.94 ± 0.21 ΔnadBC (1 × sc) 3.37 ± 0.40 ** <2 ** ΔnadBC (2 × sc) 3.6 ± 0.35 ** <2 ** BCG (1 × sc) 3.46 ± 0.19 ** <2 ** B Naive 5.56 ±0.05 4.35 ± 0.21 ΔpanCD (1 × sc) 4.99 ± 0.17 (−0.57) * 3.65 ± 0.15(−0.70) * ΔpanCD (2 × sc) 4.55 ± 0.09 (−1.01) ** 3.73 ± 0.21 (−0.62) *BCG (1 × sc) 4.71 ± 0.21 (−0.85) ** 3.35 ± 0.20 (−1.00) ** p < 0.05compared to naïve, ** p < 0.01 compared to naïve

Table 3. The attenuated M. tuberculosis ΔnadBC and ΔpanCD mutantsprotect against aerogenic challenge with M. tuberculosis Erdman. Groupsof C57BL/6 mice (5 mice per group) were vaccinated subcutaneously eitheronce or twice (6 weeks apart) with 10⁶ CFUs of mutant strains. Controlmice were vaccinated subcutaneously with 10⁶ CFUs of BCG-Pasteur. Threemonths after the initial immunization with either ΔnadBC or ΔpanCDmutant or BCG, the mice were aerogenically challenged with approximately100 CFUs of acriflavin-resistant M. tuberculosis Erdman (Ac^(r)MTB)strain as described earlier (Collins, 1985) After 28 days, thechallenged mice were sacrificed, and the lungs and spleens of individualmice were removed aseptically and homogenized separately in 5 ml ofTween 80-saline using a Seward stomacher 80 blender (Tekmar, Cincinnati,Ohio). The homogenates were diluted serially in Tween 80 saline andplated on Middlebrook 7H11 agar with or without appropriate supplementsas required. Samples from the BCG-vaccinated controls were plated on7H11 agar containing 2 mg of thiophenecarboxylic acid hydrazide (SigmaChemical Co., St Louis, Mo.) per ml to inhibit growth of any residualBCG. The CFU results were evaluated using the one-way ANOVA analysis ofthe Graph Pad InStat program. The numbers in parenthesis represent thedifferences between naïve and vaccinated organ CFUs.

In order to test the ability of the auxotrophic mutants to confer longlasting immunity, mice were challenged 7 months after an initialsubcutaneous immunization with the ΔnadBC mutant. See Table 4. Miceimmunized with ΔnadBC displayed significantly reduced numbers of thechallenge organism in the lungs and no detectable numbers in the spleencomparable to the numbers seen in the BCG vaccinated mice. The resultssuggest that the ΔnadBC vaccine strain is able to persist within themouse organs sufficiently long to mount a long lasting immunity tocontrol subsequent infection.

TABLE 4 Table 4. Immunizations with the ΔnadBC mutant confer long-termprotection against an aerosol challenge. Groups of C57BL/6 mice (5 miceper group) were vaccinated subcutaneously or intravenously either onceor twice (6 weeks apart) with 10⁶ CFUs of ΔnadBC mutant. Control micewere vaccinated subcutaneously with 10⁶ CFUs of BCG- Pasteur. Sevenmonths after the initial immunization with either ΔnadBC mutant or BCG,the mice were aerogenically challenged with approximately 50 CFUs ofacriflavin-resistant M. tuberculosis Erdman (Ac^(r)MTB) strain and thebacterial numbers at 28 days post challenge enumerated as described inTable 1. Experimental Group Lung CFUs (log₁₀) Spleen CFUs (log₁₀) Naive4.61 ± 0.07 4.07 ± 0.20 BCG 4.00 ± 0.13*  2 NAD (1 × iv) 3.28 ± 0.15**<2 NAD (2 × iv) 2.95 ± 0.14** <2 NAD (1 × sc) 4.05 ± 0.12* <2 NAD (2 ×sc) 3.94 ± 0.13* <2 *P < 0.05; **P < 0.01 by Dunnett's MultipleComparison Test

To the best of our knowledge this is the first report of any M.tuberculosis auxotrophic vaccines administered subcutaneously to conferprotection comparable to the conventional BCG vaccine strain in a mousemodel of infection. Mice vaccinated with the ΔpanCD and ΔnadBC survivedfor over one year following the aerosol challenge indicating theprotection and safety of these vaccine strains.

EXAMPLE 3 A Pantothenate Auxotroph of Mycobacterium tuberculosis isHighly Attenuated and Protects Mice Against Tuberculosis

This Example is published as Sambandamurthy et al., 2002.

Example Summary

With the advent of HIV and the widespread emergence of drug resistantstrains of Mycobacterium tuberculosis, newer control strategies in theform of a better vaccine could decrease the global incidence oftuberculosis. A desirable trait in an effective live attenuated vaccinestrain is its ability to persist within the host in a limited fashion inorder to produce important protective antigens in vivo (Kanai andYanagisawa, 1955; McKenney et al., 1999). Rationally attenuated M.tuberculosis vaccine candidates have been constructed by deleting genesrequired for growth in mice (Jackson et al., 1999; Hondalus et al.,2000; Smith et al., 2001). These candidate vaccines failed to elicitadequate protective immunity in animal models, due to their inability topersist sufficiently long within the host tissues. Here we report thatan auxotrophic mutant of M. tuberculosis defective in the de novobiosynthesis of pantothenic acid (vitamin B5) is highly attenuated inimmunocompromised SCID mice and in immunocompetent BALB/c mice. SCIDmice infected with the pantothenate auxotroph survived significantlylonger than mice infected with either BCG vaccine or virulent M.tuberculosis (250 days, vs. 77 days, vs. 35 days). Subcutaneousimmunization with this auxotroph conferred protection in C57BL/6J miceagainst an aerosol challenge with virulent M. tuberculosis, which wascomparable to that afforded by BCG vaccination. Our findings highlightthe importance of de novo pantothenate biosynthesis in limiting theintracellular survival and pathogenesis of M. tuberculosis withoutreducing its immunogenicity in vaccinated mice.

Materials and Methods.

Media and Strains. M. tuberculosis H37Rv, M. tuberculosis Erdman and M.bovis BCG Pasteur were obtained from the Trudeau Culture Collection(Saranac Lake, N.Y.) and cultured in Middlebrook 7H9 broth and 7H11 agarsupplemented with 10% OADC, 0.5% glycerol, and 0.05% Tween 80. Whenrequired, pantothenate (24 μg/ml), hygromycin (50 μg/ml) or kanamycin(25 μg/ml) was added. Stock strains were grown in Middlebrook 7H9 brothin roller bottles and harvested in mid-logarithmic growth phase, beforebeing stored in 1 ml vials at −70° C. until required.

Disruption of panCD genes in M. tuberculosis. Specialized transductionwas employed to disrupt the chromosomal copy of the panCD genes asdescribed (U.S. Pat. No. 6,271,034). Briefly, the 823 bp region upstreamto the panC gene was amplified using primers Pan1(5′-GTGCAGCGCCATCTCTCA-3′)(SEQ ID NO:9) and Pan2(5′-GTTCACCGGGATGGAACG-3′)(SEQ ID NO:10). A 716 bp region downstream tothe panD gene was amplified using primers Pan3(5′-CCCGGCTCGGTGTGGGAT-3′)(SEQ ID NO:11) and Pan4(5′-GCGCGGTATGCCCGGTAG-3′)(SEQ ID NO:12). PCR products were cloned withthe TOPO TA cloning kit (Invitrogen, CA), and sequenced. PCR productswere subsequently cloned into pJSC347, flanking a hygromycin cassette tocreate pSKV1. PacI digested pSKV1 was ligated into thetemperature-sensitive mycobacteriophage phAE159 derived from TM4 andtransduced as described earlier (Glickman et al., 2000; Raman et al.,2001). Genomic DNAs from hygromycin-resistant and pantothenate-requiringcolonies were digested with BssHII, and probed with a 716 bp downstreamregion, flanking the M. tuberculosis panCD operon to confirm thedeletion. For complementation, the M. tuberculosis pan CD operon wasamplified by PCR from genomic DNA with its putative promoter, clonedwith TA cloning kit, sequenced, and subcloned into pMV306kan, asite-specific integrating mycobacterial vector.

Animal infections. C57BL/6, BALB/cJ and BALB/c SCID mice (6-8 weeks old)were purchased from Jackson Laboratories and were infected intravenouslythrough the lateral tail vein. For time-to-death assays, BALB/c SCIDmice were infected intravenously with 1×10² CFU of M. tuberculosisH37Rv, 1×10² CFU of panCD-complemented strain, 1×10⁵ CFU of ΔpanCDmutant, or 1×10⁵ CFU of M. bovis BCG-P. For mouse organ CFU assays,BALB/cJ mice were infected with 1×10⁶ CFU of M. tuberculosis H37Rv orthe panCD-complemented strain or the ΔpanCD mutant. At appropriate timepoints, groups of 4-5 mice were sacrificed and the selected organs werehomogenized separately in PBS/0.05% Tween 80, and colonies wereenumerated on 7H11 plates grown at 37° C. for 3-4 weeks (see McKinney etal., 2000). Pathological examination was performed on tissues fixed in10% buffered formalin. The CFU results were evaluated using the one-wayANOVA analysis of the Graph Pad InStat program. All animals weremaintained in accordance with protocols approved by the Albert EinsteinCollege of Medicine Institutional Animal Care and Use Committee.

Vaccination Studies. Groups of C57BL/6 mice (5 mice per group) werevaccinated subcutaneously either once or twice (6 weeks apart) with1×10⁶ CFU of the ΔpanCD mutant strain. Control mice were vaccinatedsubcutaneously with 1×10⁶ CFU of M. bovis BCG-P. Three months after theinitial immunization with either the ΔpanCD mutant or BCG, the mice wereaerogenically challenged with approximately 50-100 CFU of M.tuberculosis Erdman strain as described earlier. At 28 days followingaerosol challenge, the challenged mice were sacrificed; the lungs andspleens of individual mice were removed aseptically and homogenizedseparately in 5 ml of Tween 80-saline using a Seward Stomacher 80blender (Tekmar, Cincinnati, Ohio). The homogenates were dilutedserially in Tween 80 saline and plated on Middlebrook 7H11 agar with orwithout appropriate supplements as required. Samples from theBCG-vaccinated controls were plated on 7H11 agar containing 2 mg/ml ofthiophene-2-carboxylic acid hydrazide (Sigma) to inhibit growth of anyresidual BCG. Results and Discussion.

Lipid biosynthesis and metabolism have been shown to play a pivotal rolein the intracellular replication and persistence of M. tuberculosis (Coxet al., 1999; Camacho et al., 1999; Glickman et al., 2000; De Voss etal., 2000; Manca et al., 2001; McKinney et al., 2000). Therefore, wesought to globally impair the ability of this bacterium to synthesizelipids. Pantothenic acid (vitamin B 5) is an essential molecule requiredfor the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP),that play important roles as acyl group carriers in fatty acidmetabolism, the tricarboxylic acid cycle, biosynthesis of polyketidesand several other reactions associated with intermediary metabolism(Jackowski, 1996). Bacteria, plants and fungi synthesize pantothenatefrom amino acid intermediates, whereas it is a nutritional requirementin higher animals (FIG. 9 a).

We constructed a double deletion mutant of M. tuberculosis in the panCand panD genes that are involved in the de novo biosynthesis ofpantothenate (FIG. 9 b,c). The ΔpanCD mutant was found to be auxotrophicfor pantothenate with no detectable reversion to prototrophy when 1×10¹⁰cells were plated on minimal medium. The growth rate of the mutant wasidentical to wild type H37Rv in broth cultures in the presence ofexogenous pantothenate (data not shown). The attenuation of the ΔpanCDmutant was assessed by infection of immunocompromised SCID mice. SCIDmice infected intravenously with H37Rv succumbed to the resultinginfection in about 5 weeks. In contrast, all mice infected with theΔpanCD mutant survived for more than 36 weeks (average, 253 days) (FIG.10 a). This attenuation is due to pantothenate auxotrophy as the fullvirulence phenotype was restored when the panCD wild type genes wereintegrated into the chromosome of the ΔpanCD mutant in single copy.Enumeration of bacterial burdens in SCID mice infected with H37Rv andthe ΔpanCD-complemented strain showed a rapid increase in bacterialnumbers in the spleen, liver and lung, until they succumbed toinfection. In contrast, mice infected with the ΔpanCD mutant showed aninitial drop in bacterial numbers in the spleen and liver followed by agradual increase in the number of viable bacteria, reaching 1×10⁶colony-forming units (CFU) by day 224 (FIG. 10B). Notably, the CFUvalues increased to 1×10⁸ in the lungs of the infected mice. The abilityof ΔpanCD-infected SCID mice to survive despite a substantial bacterialburden in their lungs emphasizes the extent of attenuation in thismutant and compares with the phenotype observed with the M. tuberculosiswhiB3 and sigH mutants described recently (Steyn et al., 2000; Kaushal,2000). Notably, SCID mice infected with bacille Calmette-Guerin-Pasteur(BCG-P) strain succumbed to infection by 83 days (Weber et al., 2000) incontrast to the prolonged survival observed in LpanCD-infected mice.

Studies in immunocompetent mice further demonstrate the attenuation ofthe ΔpanCD mutant. Survival studies showed that BALB/c mice infectedwith H37Rv succumbed to infection by day 25 (average, 21 days) and miceinfected with an identical dose of the panCD-complemented strainsuccumbed to infection between days 21 to 53 (average, 37 days).Importantly, all mice infected with 1×10⁶ CFU of the ΔpanCD mutantsurvived 375 days, when the experiment was terminated (FIG. 10C). At 3weeks post infection, in contrast to the H37Rv strain, BALB/c miceinfected with ΔpanCD mutant showed a 10-fold increase in bacterialnumbers in the lungs followed by a gradual decline in viable numbersover the next 38 weeks of infection (FIG. 10D) and the bacterial burdengradually declined in the spleen and liver throughout the course ofinfection (FIG. 10E). Histopathologic examination of the lungs from miceinfected with either H37Rv or the ΔpanCD-complemented strain, showedsevere, diffuse lobar granulomatous pneumonia (FIG. 11A,B). Thepneumonia affected more than 50% of the lung, and was pyogranulomatouswith marked necrosis in the advanced consolidated areas, particularly inthe lungs of mice challenged with H37Rv. Both of these strains causedsevere granulomatous splenitis and widespread granulomatous hepatitis.At 3 weeks post-infection with the ΔpanCD mutant, low to moderatenumbers of focal infiltrates of mononuclear cells scattered throughoutthe lung were seen (FIG. 11C). The spleen was moderately enlarged withscattered granulomas. Similarly, the liver showed numerous focalgranulomas. At 24 weeks post-infection, consistent with the bacterialnumbers, histological examination of the lungs from mice infected withthe ΔpanCD mutant showed only occasional focal mild infiltrations,predominately lymphocytic (FIG. 11D). The spleen showed only mildhistiocytic hyperplasia and there were fewer, focal, predominatelylymphocytic accumulations in the liver.

The mechanisms that allow the persistence of the ΔpanCD mutant bacteriafor over 8 months in the SCID mouse model remain unclear. We speculatethe functional role of an unidentified permease in transporting adequateamount of pantothenate in the ΔpanCD mutant that allows its persistencebut not the ability to cause disease. A pantothenate permease thattransports pantothenate have been described in Plasmodium falciparum andEscherichia coli (Saliba and Kirk, 2001; Jackowski and Alix, 1990). Inthe lungs of immunocompetent mice, an initial growth of the ΔpanCDmutant during the first 3 weeks is followed by a steady decline inbacterial numbers following the onset of an adaptive immune response.The intracellular lifestyle of M. tuberculosis poses significantchallenges to the bacterium in acquiring essential nutrients.Pantothenic acid or its derivatives have been shown to confer resistanceto oxidative stress (Slyshenkov et al., 1996) and lack of pantothenatebiosynthesis in the ΔpanCD mutant may render it more susceptible to suchadverse effects. Likewise, a pantothenate kinase (panK) mutant ofDrosophila was shown to display membrane defects and improper mitosisand meiosis due to decreased phospholipid biosynthesis (Afshar et al.,2001). Therefore, it is plausible that the pantothenate salvage pathwayis inadequate in restoring full virulence of the ΔpanCD mutant in theabsence of a functional de novo biosynthetic pathway.

As a test of vaccine potential, immunized mice were challenged withvirulent M. tuberculosis Erdman by the aerosol route (Collins, 1985).Following subcutaneous immunization, the ΔpanCD mutant could not bedetected in the spleens or lungs of mice at 8 and 12 weeks. In the naivecontrols, the bacterial CFU values increased 10.000-fold in the lungduring the first month after challenge. Similarly, substantialdissemination and growth in the spleen was detected within one month ofthe challenge in naive controls. In contrast, mice immunized with singleor double doses of the ΔpanCD mutant displayed statistically significantreductions (P<0.05) in lung and spleen CFU values relative to naivecontrols. Mice vaccinated with BCG showed similar reduction in organbacterial burdens compared to the nonimmunized controls (FIG. 11E,F). Inthese aerogenic challenge studies, no significant differences weredetected in the lung and spleen CFU values for mice vaccinated witheither the ΔpanCD mutant strain or with BCG. At 28 days after theaerogenic challenge with virulent M. tuberculosis, histopathologicalexamination of lungs of ΔpanCD immunized mice revealed a less severeinfection relative to the unvaccinated control mice. In controls, severebronchitis, moderate pneumonia, and spread of the infection to theadjacent lung parenchyma was observed. By comparison, the ΔpanCDvaccinated mice had milder bronchitis and smaller areas of mildinterstitial pneumonitis, with localized areas of granulomatouspneumonia in some mice. Importantly, no lung pathology was detected invaccinated mice at the time of challenge (data not shown). Two groups ofmice that were vaccinated with one or two doses of the ΔpanCD mutant andthen challenged with M. tuberculosis Erdman were active and healthy formore than one year following the virulent challenge. Histopathologicalanalysis of lung sections from these mice showed only mild inflammationand fibrosis despite the chronic infection.

By creating a M. tuberculosis strain that is defective in pantothenatebiosynthesis, we have taken a critical step in the rational developmentof an attenuated M. tuberculosis vaccine strain. We have shown that afunctional pantothenate biosynthetic pathway, which is required for thesynthesis of complex mycobacterial lipids, is essential for thevirulence of M. tuberculosis. Although the precise mechanism of thereduced virulence is unclear, it is reasonable to speculate that thiscould be due to reduced synthesis of toxic polyketides and secretedlipids or a general slow down of metabolism. Tubercle bacilli lackingthe two genes required to synthesize pantothenate failed to revert andwere highly attenuated and less virulent than BCG vaccine when tested inthe rigorous SCID mouse model of infection. Despite the reducedvirulence associated with the deletion of the panCD genes, these vitaminauxotrophs remain persistent in vivo as shown by their ability tosurvive for at least eight months in immunocompetent mice. Thepersistence of this mutant strain undoubtedly contributes to thesubstantial immunogenicity seen in the mouse tuberculous challengemodel. Overall, the ΔpanCD mutant has many of the characteristicsnecessary for a live vaccine candidate strain: it is attenuated by anon-reverting mutation and essentially avirulent while being persistentand immunogenic. Given the genetic differences between M. bovis and M.tuberculosis (Behr et al., 1999), one would predict that a rationallyattenuated M. tuberculosis strain would have a more relevant repertoireof species-specific antigens and thus should elicit, in humans, moreeffective protective immune responses against tuberculous infectionsthan BCG.

EXAMPLE 4 The Primary Mechanism of Attenuation of BCG is a Loss ofInvasiveness Due to Host Cell Lysis

Example Summary.

Tuberculosis remains a leading cause of death worldwide, despite theavailability of effective chemotherapy and a vaccine. BCG, thetuberculosis vaccine, is an attenuated mutant of M. bovis that wasisolated following serial subcultivations, yet the basis for thisattenuation has never been elucidated. A single region (RD1), deleted inall BCG substrains, was deleted from virulent M. bovis and M.tuberculosis strains and the resulting three ΔRD1 mutants weresignificantly attenuated for virulence in both immunocompromised andimmunocompetent mice. Like BCG, M. tuberculosis ΔRD1 mutants protectmice against aerosolized M. tuberculosis challenge and these mutantsalso consistently display altered colonial morphotypes. Interestingly,the ΔRD1 mutants failed to cause necrosis, via lysis, of pneumocytes, aphenotype that had been previously used to distinguish virulent M.tuberculosis from BCG. We conclude that the primary attenuatingmechanism of BCG is the loss of cytolytic activity, resulting in reducedinvasiveness.

Introduction.

BCG (bacille Calmette and Guerin), was first isolated from M. bovisfollowing serial subculturing of M. bovis in 1908 (Calmette and Guerin,1909). Drs. Calmette and Guerin set out to test the hypothesis that abovine tubercle bacillus could transmit pulmonary tuberculosis followingoral administration (Calmette and Guerin, 1905; Gheorghiu, 1996) anddeveloped a medium containing beef bile that enabled the preparation offine homogenous bacillary quspensions. After the 39th passage, thestrain was found to be unable to kill experimental animals (Calmette andGuerin, 1909). Between 1908 and 1921, the strain showed no reversion tovirulence after 230 passages on bile potato medium (Gheorghiu, 1996),which is consistent with the attenuating mutation being a deletionmutation. In the animal studies that followed, BCG was shown to beattenuated, but it also protected animals receiving a lethal challengeof virulent tubercle bacilli (Calmette and Guerin, 1920). BCG was firstused as a vaccine against tuberculosis in a child in 1921 (Weill-Halleand Turpin, 1925). From 1921 to 1927, BCG was shown to have protectiveefficacy against TB in a study on children (Id.; Calmette and Plotz,1929) and was adopted by the League of Nations in 1928 for widespreaduse in the prevention of tuberculosis. By the 1950's, after a series ofclinical trials, the WHO was encouraging widespread use of BCG vaccinethroughout the world (Fine and Rodrigues, 1990). Although an estimated 3billion doses have been used to vaccinate the human population againsttuberculosis; the mechanism that causes BCG's attenuation remainsunknown.

Mahairas et al. (1996) first compared the genomic sequences of BCG andM. bovis using subtractive hybridization and found that there were threeRegions of Difference (designated RD1, RD2, and RD3) present in thegenome of M. bovis, but missing in BCG. Behr et al. (Behr et al., 1999)and others (Gordon et al., 2001) later identified 16 large deletions,including RD1 to RD3, present in the BCG genome but absent in M.tuberculosis. Eleven of these 16 deletions were unique to M. bovis,while the remaining 5 deletions were unique to BCG. One of these 5deletions, designated RD1 (9454 bp), was absent from all of the BCGsubstrains currently used as TB vaccines worldwide and it was concludedthat the deletion of RD1 appeared to have occurred very early during thedevelopment of BCG, probably prior to 1921 (Behr et al., 1999). It isreasonable to hypothesize that RD1 was the primary attenuating mutationfirst isolated by Calmette and Guerin to generate BCG from M. bovis.Attempts to restore virulence to BCG with RD1-complementing clones havebeen unsuccessful (Mahairas et al., 1996).

Results

RD1 deletions of M. bovis and M. tuberculosis are attenuated forvirulence in immunocompromised mice. To test if RD1 is essential forvirulence in M. bovis and M. tuberculosis, it was necessary to deletethe RD1 (FIG. 1 a) from virulent strains, demonstrate loss of virulence,and then restore virulence by complementation with the RD1 DNA. Sincethe original M. bovis parent of BCG was lost in World War I (Grange etal., 1983), we initiated studies with virulent M. bovis Ravenel and avariety of virulent M. tuberculosis strains. Despite success ingenerating an unmarked deletion mutant of RD1 in M. tuberculosis with aplasmid transformation system^(1,2), over 100 independenttransformations failed to yield an RD1 deletion in M. bovis. As analternative strategy, specialized transduction (Bardarov et al., 2002)³was successfully used to generate RD1 deletion mutants not only in M.bovis Ravenel, but also the H37Rv, Erdman, and CDC1551 strains of M.tuberculosis (FIG. 12). This deletion represents the largest deletionmutation generated by a targeted disruption in M. tuberculosis or M.bovis made to date and demonstrates the utility of the specializedtransduction system. Moreover, since the parental specializedtransducing phage has been shown to infect over 500 clinical M.tuberculosis isolates (Jacobs et al., 1987), it should be possible tointroduce the RD1 deletion into any M. tuberculosis or M. bovis isolateof interest.

To determine if the RD1 deletion causes an attenuating phenotype in M.bovis and M. tuberculosis, the M. tuberculosis H37Rv ΔRD1 was inoculatedintravenously into immunocompromised mice possessing the SCID (severecombined immunodeficiency) mutation. Groups of ten mice were injectedintravenously with either 2×10⁶ wild type or ΔRD1 strain of M.tuberculosis and M. bovis, and three mice per group were sacrificed 24hours later to verify the inoculation doses. All of the SCID miceinfected with the parental M. tuberculosis or M. bovis strain diedwithin 14 to 16 days post-infection (FIG. 12A). In contrast, the SCIDmice infected with equal doses of the ΔRD1 strains of M. tuberculosis orM. bovis were all alive at 25 to 41 days post-infection, demonstrating ahighly significant attenuation of the virulence of both strains. It isimportant to note that BCG-Pasteur kills SCID mice approximately 70 dayspost-infection (FIG. 13B), suggesting that BCG substrains have acquiredadditional attenuating mutations which are consistent with the deletionanalysis of BCG strains (Behr et al., 1999) and the previous failures torestore virulence with the RD1 region (Mahairas et al., 1996).

To prove that the attenuation of virulence was due to the RD1 deletion,the M. tuberculosis ΔRD1 was transformed with an integrating cosmid,2F9, containing the RD1 region from M. tuberculosis H37Rv⁴. SCID micewere infected as described above and the attenuation for virulence wasrestored to the parental virulent phenotype (FIG. 13B). These resultsstrongly suggest that the genes deleted from the RD1 region contributeto the virulence phenotype.

The M. tuberculosis ΔRD1 is highly attenuated in immunocompetent BALB/cmice. The virulence of the M. tuberculosis ΔRD1 mutant was furtherassessed by intravenous inoculation of immunocompetent BALB/c mice.While the virulent parent M. tuberculosis strain killed the BALB/c micein 10 to 17 weeks post-infections, 100% of mice were alive at 48 weeksand 43 weeks post-infections in two independent experiments (FIG. 13C).

While infection with BCG and M. tuberculosis ΔRD1 yielded similarsurvival results in BALB/c mice, there were substantial differences inthe growth kinetics in mice. BCG grew in a limited fashion in lungs,liver and spleen in BALB/c mice during the 22 weeks of the experiment(FIGS. 4 b-d). In contrast, the M. tuberculosis ΔRD1 strain grew in afashion indistinguishable from the parental M. tuberculosis H37Rv in allmouse organs for the first 8 weeks. Thereafter, mice infected with theparental M. tuberculosis failed to contain the infection leading tomortality. Strikingly, mice infected with the M. tuberculosis ΔRD1showed a definite control over infection resulting in significantlyprolonged survival of mice (FIG. 4 b-d).

Histopathological examination further demonstrated that the mutant wasattenuated in virulence compared to the parent strain H37Rv (FIG. 5d-f). In contrast to the rapidly progressive infection with the parentstrain, the lung lesions caused by the mutant were maximal in miceexamined at 8 weeks post-infection. Consolidating granulomatouspneumonia involved an estimated 25-30% of the lung in these mice.Numerous organisms were demonstrated by acid fast staining. Thepneumonia subsequently underwent partial resolution. By 14 weeks, andagain, at 22 weeks post-infection, the lungs showed peribronchial andperivascular inflammatory cell accumulations and focal, generallynon-confluent, granulomas now with a prominent lymphocytes infiltration.The numbers of acid fast bacilli were reduced. Liver lesions consistedof low numbers of scattered granulomas. Spleens were smaller, withpersistent granulomas in the red pulp. Mice infected with M. bovis BCGshowed mild lesions in the lung, liver and spleen at all time points(FIG. 5 g-i). At longer time intervals post-infection the lesions werefewer in number, and smaller with prominent lymphocytic infiltrations.At 14 weeks post-infection, M. bovis BCG was below the level ofdetection by acid fast staining. In summary, whereas M. tuberculosisΔRD1 initially grew in a manner similar to the parental M. tuberculosisH37Rv, this RD1 mutant was limited in the extent of spread of infection,particularly in the lung. This contrasts the extensive and severe damagecaused by the parent strain. The subsequent resolving granulomas,localization of the lesions and changes in the composition of theinflammatory cell infiltrations suggested that the mice mounted aneffective immune response to combat M. tuberculosis ΔRD1 infection andthereby reduced the numbers of viable organisms.

Early BCG properties: Altered colonial morphotypes and long-termimmunogenicity.

While frozen stocks of the original BCG strain do not exist, writtenrecords do exist describing the early BCG strains, as Dr. Calmette sentthe strains to many laboratories. In a study published in 1929, Petroffand colleagues reported that BCG displayed two distinct colony types(Petroff et al., 1929). One morphotype was a smooth (S) phenotype thatwas flat and corded (like the parental virulent strain) and the secondwas a rough and raised (R) phenotype. The M. tuberculosis ΔRD1 mutantwas generated independently four times and consistently yielded a 20 to50% mixture of two colonial morphotypes on Middlebrook medium withoutTween 80 (FIG. 3 b). The distinction of these two types of morphologycould be noted even when the colonies were less than two weeks old, asthe rough colonies were constricted and elevated with only a smallportion of the base of the colony attached to the agar, while the smoothcolonies tended to be flattened and spread out. When colonies grewolder, e.g. 6 weeks old, the rough colonies remained more constrictedcompared to those of smooth colonies. The rough colonies exhibited largefolds on the surface (FIG. 3 f-g), as compared to those of the smoothcolonies that exhibited small wrinkles (FIG. 3 e).

The generation of two distinct colonial morphotypes must be a phenotypicchange induced by the deletion of RD1. The morphotypes could not becloned, as repeated subculturing of either the R or S colonies continuedto yield both colonial morphotypes. Moreover, Southern analysis of R andS colonies revealed each morphotype had the same RD1-deleted genotype(FIG. 3 d). Furthermore, complementation of M. tuberculosis ΔRD1 withthe RD1 region restored the mutant phenotype back to the homogenousparental S phenotype (FIG. 3 a-c). These results suggest that thevariable morphotypes resulted directly from the RD1 deletion. It cantherefore be postulated that a regulator of colonial morphology isaffected by one or more of the deleted genes.

One of the hallmark characteristics of BCG is its ability to provideprotection against aerosolized challenge with virulent M. tuberculosis.To test the potential of M. tuberculosis ΔRD1 to immunize and protectmice against tuberculous challenge, we used the model of subcutaneousimmunization of C57BL/6 mice followed by an aerogenic challenge withvirulent M. tuberculosis (McGuire et al., 2002). Groups of mice werevaccinated subcutaneously with either 1×10⁶ BCG 9 or 1×10⁶ M.tuberculosis ΔRD1. Eight months following vaccination, the mice were allhealthy, thereby demonstrating attenuation in a third mouse strain.Vaccinated and unvaccinated mice were aerogenically challenged with 200CFU of the acriflavin-resistant strain of M. tuberculosis Erdman.Twenty-eight days after the challenge, the mice were sacrificed and thebacterial burden in the lungs and spleens were determined (see Table 5).Naive mice served as controls. While the acriflavin-resistant M.tuberculosis grew to 6.61±0.13 (log¹⁰ CFU) in lungs of naive mice, boththe BCG-vaccinated and M. tuberculosis ΔRD1-vaccinated mice exhibitedgreater than 1 log protection in lungs with CFU values of 5.07±0.10(p<0.001 relative to controls) and 5.11±0.14 (p<0.001), respectively,detected at the four week time point. The M. tuberculosis ΔRD1 alsoprotected against hematogenous spread; CFU values in the spleen were5.26±0.11 for the controls, 4.00±0.33 (p<0.01) for the M. tuberculosisΔRD1 immunized mice, and 3.85±0.17(p<0.01) for the BCG vaccinatedanimals. Thus, the M. tuberculosis ΔRD1 shares long-term immunogenicitylike BCG.

TABLE 5 Bacterial burden of virulent M. tuberculosis in uninoculatedmice and mice inoculated with BCG and H37Rv ΔRD1. Vaccination strainLung (log₁₀CFU) Spleen (log10CFU) — 6.61 ± 0.13 5.26 ± 0.11 BCG 5.07 ±0.10*** 3.85 ± 0.17** H37Rv ΔRD1 5.11 ± 0.14*** 4.00 ± 0.33** **p <0.01; ***p < 0.001.Discussion

BCG is a mutant of M. bovis that was isolated over 94 years ago andcharacterized for its attenuation for virulence in animals. For over 80years, BCG has been used as a tuberculosis vaccine having been given to3 billion humans. It is currently the only anti-tuberculous vaccineavailable for use in humans, yet its precise attenuating mutations andmechanisms of attenuation have never been determined. Previous studieshad identified regions of the M. bovis chromosome that were absent fromBCG, but present in virulent M. bovis and M. tuberculosis strains(Mahairas et al., 1996; Gordon et al., 2001). An elegant microarrayanalysis has also demonstrated that there was only one deletion commonto all BCG strains; the authors hypothesized this was the primaryattenuating mutation in the original BCG strain isolated by Drs.Calmette and Guerin (Behr et al., 1999).

Using a combination of targeted deletion mutagenesis, virulence assays,and complementation analysis, we have been able to unambiguously provethat RD1 is required for virulence for M. tuberculosis, and by analogyfor M. bovis, for the first time. Moreover, the combination ofphenotypes associated with the early BCG strains: i) the attenuation forvirulence, ii) the altered colonial morphotypes, and iii) the ability toconfer long-term immunogenicity in animals allow us to conclude that theRD1 deletion was the primary attenuating mutation in the original BCGisolate.

With regards to the ΔRD1 mutant histology, at 22 weeks post infection,it was noted that the mutant was limited in the extent of the spread ofinfection, in contrast to the extensive damage caused by the parentalstrain. Interestingly, Pethe et al. (2001) determined that M.tuberculosis needs to bind and/or invade epithelial cells in order todisseminate and cause widespread destruction of the lung, whilst anotherstudy reported that pulmonary M cells can act as a portal of entry tothe lung for the tubercle bacilli (Teitelbaum, 1999). In relation to invitro analyses, studies utilizing a model of the alveolar barrier,consisting of pneumocytes and monocytes, described how M. tuberculosisinfection of the pneumocytes resulted in cytolysis, which disrupted thebarrier and allowed more efficient translocation of intracellularbacilli (Bermudez et al., 2002).

Notes

¹The following four primers were used to amplify upstream and downstreamflanking sequences (UFS and DFS, respectively) for the construction ofthe RD1 deletion mutants. UFS was amplified using TH201:GGGGGCGCACCTCAAACC (SEQ ID NO:5) and TH202: ATGTGCCAATCGTCGACCAGAA (SEQID NO:6). DFS was amplified using TH203: CACCCAGCCGCCCGGAT (SEQ IDNO:7), and TH204: TTCCTGATGCCGCCGTCTGA (SEQ ID NO:8). Recognitionsequences for different restriction enzymes were included at the ends ofeach primer to enable easier manipulation.

²The unmarked deletion mutant of M. tuberculosis H37Rv, mc²4002, wasgenerated by transformation using a sacB counterselection (Snapper etal., 1988; Pelicic et al., 1996; Pavelka et al., 1999). Specifically,the plasmid pJH508 was created by first cloning UFS into KpnI and XbaIsites, then cloning DFS into EcoRI and HindIII sites of pJH12, apMV261-derived E. coli-Mycobacteria shuttle plasmid, to create pJH506 inwhich UFS and DFS flanked a green fluorescent protein gene (GFPuv,Clonetech) whose expression was driven by the M. leprae 18 Kd promoter.The UFS-gfp-DFS cassette was sub-cloned into the EcoRV site of plasmidpYUB657 to create pJH508. The first homologous recombination involvedthe identification of hygromycin resistant colonies, resulting from thetransformation of M. tuberculosis with pJH508. Southern analysis of theNcoI-digested DNA isolated from hygromycin resistant colonies probedwith UFS or DFS, confirmed the presence of a single copy of pJH508inserted into the M. tuberculosis genome. The transformant (mc²4000)identified was then grown in 7H9 broth to saturation, to allow thesecond homologous recombination to occur, resulting in recombinants thatcould be selected by plating the culture on 7H10 plates, supplementedwith 3% sucrose. Both Southern analysis and PCR of the DNA isolated fromsucrose resistant colonies confirmed the RD1 deletion.

³Specialized transduction is a mycobacteriophage-based method for thedelivery of homologous DNA constructs using conditionally replicatingshuttle phasmids (Jacobs et al., 1987; Bardarov et al., 1997; Carriereet al., 1997) has been used successfully for M. tuberculosis (Glickmanet al., 2000, 2001; Raman et al., 2001). Specifically, a transducingphage phAEKO1 was constructed by inserting UFS and DFS into pJSC347,flanking a hygromycin cassette, to create pJH313. pJH313 was digestedwith PacI and ligated to phAE159, a temperature-sensitivemycobacteriophage derived from TM4. The transduction was performed bygrowing M. tuberculosis to an O.D.₆₀₀ of 1.0, washing twice with MPbuffer (50 mM Tris pH 7.6, 150 mM NaCl, 10 mM MgCL₂, 2 mM CaCl₂),resuspending into an equal volume of MP buffer and mixing with thetransducing phage phAEKO1 at an MOI of 10. The mixtures were incubatedat 37° C. overnight, then plated on 7H10 plates supplemented withhygromycin at 50 μg/ml. Hygromycin resistant colonies were analyzed byPCR and Southern analysis, as described above, to confirm the deletionof RD1.

⁴Complementation analyses was performed using the integration proficientcosmids (Skjot et al., 2000; van Pinxteren et al., 2000a) pYUB412 madeby S. Bardarov, a library made by F. Bange, and cosmid identified andgenerously provided by S. T. Cole.

EXAMPLE 5 Vaccine Efficacy of a Lysine Auxotroph of M. tuberculosis

In this Example, we describe the in vivo growth phenotype and vaccineefficacy of a lysine auxotrophic mutant of Mycobacterium tuberculosisstrain H37Rv. An immunization experiment using the mouse model with anaerosol challenge showed that two doses of the M. tuberculosis mutantwere required to generate protection equivalent to that of the BCGvaccine.

Despite the existence of anti-microbial drugs and a widely used vaccine,Mycobacterium tuberculosis remains the primary cause of adult death dueto a bacterial agent (Dolin et al., 1994). The emergence of multi-drugresistant strains of M. tuberculosis, the variable efficacy of thecurrent vaccine, the bacille-Calmette and Geurin (BCG), and the HfVpandemic have all contributed to a growing global tuberculosis problem.

Several studies have described the development of attenuated auxotrophicstrains of BCG and/or M. tuberculosis (Guleria et al., 1996); Hondaluset al., 2000; Jackson et al., 1999; Smith et al., 2001). All of thesestudies utilized single immunization protocols and demonstrateddifferences in the protective responses thus elicited. In this study, wedescribe the in vivo growth characteristics of a previously describedlysine auxotroph of M. tuberculosis H37Rv (Pavelka and Jacobs, 1999),and evaluate the vaccine potential of this mutant by a multipleimmunization protocol in a mouse model of the human disease, using anaerosol challenge.

Clearance of the M. tuberculosis lysine auxotroph in SCID mice. FemaleSCID mice were bred at the animal facility of the Albert EinsteinCollege of Medicine. The animals were maintained under barrierconditions and fed sterilized commercial mouse chow and water adlibitum. The M. tuberculosis strains Erdman, mc²3026 (ΔlysA::res) (Id.),and mc²3026 bearing pYUB651 (expressing the wild-type lysA gene) weregrown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween-80,O.2% glycerol, 1×ADS (0.5% bovine serum albumin, fraction V (Roche);0.2% dextrose; and 0.85% NaCl) or on Middlebrook 7H10 or 7H11 solidmedium (Difco) supplemented with 0.2% glycerol and 10% OADC (BectonDickinson). Culture media for the lysine auxotroph were supplementedwith 1 mg/ml of L-lysine (for both liquid and solid media), and 0.05%Tween-80 was also added to solid medium. Liquid cultures were grown in490 cm² roller bottles (Corning) at 4-6 rpm. Plates were incubated for3-6 weeks in plate cans. All cultures were incubated at 37° C.

Titered frozen stocks of the bacteria were thawed and dilutedappropriately in phosphate buffered saline containing 0.05% Tween-80(PBST). The bacterial suspensions were plated at the time of injectionto confirm the number of viable bacteria. Intravenous injections weregiven via a lateral tail vein. At various time points post-injection (24hours, then once weekly), 3 mice were sacrificed, and the lungs, liver,and spleen removed and homogenized separately in PBST using a Stomacher80 (Tekmar, Cincinnati, Ohio). The homogenates were diluted in PBST andplated to determine the number of CFU/organ. Note that mice weresacrificed at 24 hours post-injection in order to compare the bacterialcolony forming units recovered from the mice with the colony formingunits in the suspensions at the time of injection. Thus the bacterialcounts reported at time zero actually represent the viable bacteriarecovered from the mice at 24-hours post-injection.

The lysine auxotrophic strain was cleared from and did not appear togrow in the examined organs of the SCID mice, while the complementedstrain multiplied extensively (FIG. 14). Interestingly, the auxotrophicinoculum was cleared from the spleens and lungs but persisted somewhatlonger in the liver (FIG. 14B). The mice receiving the complemented M.tuberculosis mutant died within three weeks of challenge, while the micegiven the auxotrophic M. tuberculosis mutant did not display any grossorgan pathology and survived for at least the duration of theexperiment.

Two immunizations with the M. tuberculosis lysine auxotroph mc²3026 arerequired to match the efficacy of vaccination with BCG-Pasteur. Wetested the vaccine potential of the lysine auxotroph mc²3026 in themouse model by means of a virulent aerosol challenge. Female,pathogen-free C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me.) werevaccinated intravenously with ca. 1×10⁶ CFU of the M. tuberculosislysine auxotroph or BCG-Pasteur suspended in 0.2 ml PBST. Micevaccinated with mc²3026 were revaccinated at 4 week intervals and thenumber of viable organisms in the lungs and spleens determined weeklythroughout the vaccination period, as described above for the SCID mouseexperiments. Five mice were examined at each time point.

Immunized mice were challenged 3 months after the initial vaccination. Afrozen aliquot of a M. tuberculosis Erdman stock was thawed and dilutedin PBST to ca. 1×10⁶ CFU/ml and 10 ml was introduced into the nebulizerof a Middlebrook aerosol chamber (Glas-Col, Terre Haute, Ind.). The micewere exposed to the infectious aerosol for 30 minutes, inhaling 50-100CFU into their lungs over this period. Five mice were sacrificedimmediately following the challenge period and the lung homogenates wereplated to check the amount of the challenge inoculum actually reachingthe lungs. Groups of vaccinated and control mice were sacrificed 14, 28,and 42 days later and the lung and spleen homogenates plated todetermine the number of viable colony forming units of M. tuberculosisErdman present. Data were analyzed using the Student's t-test and ananalysis of variance between several independent means, using the InStat Statistics program (GraphPad Software, San Diego).

A preliminary experiment demonstrated that a single intravenousimmunization of immunocompetent C57BL/6 mice with the M. tuberculosismutant did not generate a significant protective response to thesubsequent aerosol challenge with virulent M. tuberculosis Erdman. Inthat experiment, the M. tuberculosis auxotroph was rapidly cleared fromthe mice (FIG. 15A), and the single immunization with the auxotroph wasinsufficient to reduce the bacterial burden in the lungs and spleensrelative to a single immunization with BCG (FIG. 15B).

The failure of the auxotroph to confer protection might have been due tothe inability of the mutant to persist long enough, or to synthesizeenough antigen to induce an immune response that could significantlyrestrict the growth of the challenge organisms. One way to circumventthis problem is to give multiple doses of vaccine (Collins, 1991;Homchampa et al., 1992). To this end, mice were intravenously immunizedtwo or three times at four-week intervals with the M. tuberculosislysine auxotroph. In both cases, the vaccine strain was cleared from thelungs and spleens of all the mice at rates similar to that seen with thesingle immunization experiment (FIG. 15A). Three months after the firstimmunization the mice were challenged with M. tuberculosis Erdman by theaerosol route and the bacterial counts in the lungs and spleens weredetermined and compared to a BCG-Pasteur immunized control, as well asthe sham immunized controls. As seen in FIG. 15C, double immunizationwith the M. tuberculosis lysine auxotroph induced a protective responsethat was equivalent to that of the BCG control. The reduction in countsin the lung and spleen was equivalent to a 100-fold reduction inbacterial counts compared to the unvaccinated control (FIG. 15C). Theresults from the triple immunization experiment were essentially similaras those from the double immunization experiment described above (datanot shown). Furthermore, mice that were immunized with three doses ofthe M. tuberculosis lysine auxotroph and challenged with virulent M.tuberculosis Erdman survived at least as long as the BCG-immunizedcontrol mice (FIG. 16).

Several studies have described the development and vaccine efficacy ofattenuated mutant strains of M. tuberculosis (Jackson et al., 1999;Hondalus et al., 2000; Smith et al., 2001). The first study reportedthat a purine auxotroph of M. tuberculosis was unable to grow inmacrophages and was attenuated for growth in both mice and guinea pigs(Jackson et al., 1999). A guinea pig vaccination experiment determinedthat a single immunization with the auxotroph allowed the animals torestrict the growth of virulent M. tuberculosis in the lungs as well asa single immunization with wild-type BCG, following aerosol challenge.However, the reduction in growth of the challenge organism in the spleenafforded by the auxotroph was not as extensive as that afforded by BCG.Another study reported that a leucine auxotroph of M. tuberculosisErdman cannot grow in macrophages and is avirulent to immunocompromisedSCID mice (Hondalus et al., 2000). Immunocompetent mice vaccinated oncewith a M. tuberculosis leucine mutant did not significantly restrict thegrowth of the virulent challenge organism in the lungs or spleen as muchas the control mice vaccinated with BCG (Id.). However, the miceimmunized with the leucine auxotroph survived as long as the BCGimmunized controls and exhibited a decreased histopathology relative tothat seen in the non-immunized controls (Id.) A third study showed thatM. tuberculosis proline and tryptophan auxotrophs were attenuated and asingle immunization of mice with either of these mutants affordedprotection against an intravenous challenge with virulent M.tuberculosis, comparable to that for BCG, as indicated by the meansurvival times (Smith et al., 2001). In those experiments, miceimmunized with pro or trp mutants could restrict the growth of thechallenge organisms to the same extent as mice immunized with BCG,although the magnitude of protection in either case (M. tuberculosisauxotrophs or BCG) was not as extensive as that seen in the otherstudies (Id.).

In the present study we have demonstrated that a single immunization ofmice with the avirulent M. tuberculosis lysine auxotroph did notgenerate an immune response capable of significantly restricting thegrowth of virulent M. tuberculosis Erdman following an aerogenicchallenge. However, administration of a second or a third dose of thisvaccine increased protection substantially, as measured by the number ofviable bacteria per organ, to a level similar to that achieved withsingle dose of BCG-Pasteur. This level of protection did not seem to begreatly increased by a third dose of vaccine, although the triplyimmunized mice survived as long as the control mice immunized with asingle dose of BCG-Pasteur. Mice that were immunized twice were notfollowed to determine mean survival time, but comparing the growthcurves of the challenge bacteria following the double and tripleimmunizations, it seems likely that the survival time for the doublyimmunized mice would be much the same as that for the triple-immunizedmice.

The previous studies using M. tuberculosis auxotrophs as vaccine strainsshowed substantial variations in their effectiveness. This variabilityis likely to be due to a number of factors, including the different M.tuberculosis background strains used to construct the mutants, differentmouse strains used in the various protection studies, and the differentchallenge organisms and challenge routes used. There was alsoconsiderable variation in the protective efficacy of the differentvaccines compared to that observed in controls using BCG immunization.These differences pose a number of questions concerning the bestindicators of protection, especially in the long term. Should viablebacterial counts or survival be the primary indicator of protection orshould both be given equal weight? The results of this study indicatethat more than one immunization with a M. tuberculosis lysine auxotrophdid generate a significant protective response as indicated by bothcriteria. We believe it is important that multiple immunizationprotocols be considered in the further development of attenuated M.tuberculosis strains as potential human vaccines.

This is the first study demonstrating that a multiple immunizationprotocol using an auxotroph of M. tuberculosis can protect against ahighly virulent aerosol challenge compared to that seen for BCG. SinceBCG vaccines have shown variable efficacy when tested in humans, anauxotrophic M. tuberculosis vaccine might represent an attractivebooster vaccine with which to augment childhood BCG immunization.

EXAMPLE 6 Mutants of Mycobacterium tuberculosis Having Two AttenuatingMutations are Safe and Provide Protection in Mammals Lacking CD4⁺Lymphocytes

The experiments described in this Example employ materials and methodsdescribed in the other Examples.

Construction and characterization of M. tuberculosis ΔRD1ΔpanCD(mc²6030). A pantothenate auxotroph of M. tuberculosis ΔRD1 wasgenerated by specialized transduction and the strain designated mc²6030.No CFU were detected on 7H11 when 5×10¹⁰ CFU were plated (repeatedtwice), suggesting the reversion frequency to be below 10⁻¹¹.

SCID mice infected with 1×10² CFU H37Rv succumbed to infection in 6weeks, whereas the mice infected with 1×10⁶ mc²6030 survivedsignificantly longer with more than 75% of mice surviving for more than300 days (FIG. 17A). Bacteria isolated from mc²6030-infected mice beforethey died were all auxotrophs, confirming that there were no revertantsunder in vivo conditions. In order to assess the safety of mc²6030 inimmunocompetent BALB/c mice, we infected mice intravenously with 1×10⁶mc²6030 or 1×10⁶ of wild-type H37Rv. All mice infected with H37Rvsuccumbed to infection by 150 days, whereas mice infected with mc²6030survived for more than 300 days (FIG. 17B). In an effort to understandthe role of immune responses in controlling infection with thepantothenate mutants, we infected immunocompetent C57Bl/6 with 1×10⁶ CFUof mc²6001 (ΔRD1), mc²6004 (complementing strain), mc²6030 (ΔRD1ΔpanCD)or wild-type H37Rv. Mice infected with H37Rv and mc²6004 showedprogressive growth in all the three organs, whereas mice infected withmc²6030 showed a drop in growth during the first 3 weeks in the lungsand spleen (FIG. 18). Following 3 weeks of infection, the growth patternof both mc²6001 and mc²6030 were identical in the spleen and lungs. Miceimmunized subcutaneously with one or two doses of mc²6030 demonstratedprotection against aerosol challenge with virulent M. tuberculosis,which was comparable to the protection afforded by BCG vaccination(Table 6). No pantothenate auxotrophs were recovered from spleen orlungs of mice at 1, 2 or 3 months following subcutaneous immunization.

TABLE 6 Bacterial burden of virulent M. tuberculosis in uninoculatedmice and mice inoculated with BCG or one or two doses of ΔRD1ΔpanCD.Experimental Group Lung CFUs (log₁₀) Spleen CFUs (log10) Naive 5.99 ±0.09 4.94 ± 0.06 ΔRD1ΔpanCD (1 dose) sc 5.22 ± 0.10* 4.04 ± 0.15*ΔRD1ΔpanCD (2 doses) sc 4.86 ± 0.14** 3.58 ± 0.11** BCG (1 dose) sc 4.79± 0.19** 3.73 ± 0.27** *p < 0.01 relative to controls; **p < 0.001relative to controls

Construction and characterization of M. tuberculosis ΔlysAΔpanCD(mc²6020). A pantothenate auxotroph of M. tuberculosis ΔlysA wasgenerated by specialized transduction and the strain designated mc²6020.No CFU were detected on 7H11 when 5×10¹⁰ CFU were plated, suggesting thereversion frequency to be below 10⁻¹¹. This double mutant is auxotrophicfor both lysine and pantothenate. SCID mice infected with 1×10² CFUH37Rv succumbed to infection in 6 weeks, whereas the mice infected with1×10⁶ mc²6020 survived for more than 400 days with no mortality. Inorder to assess the safety and growth kinetics of mc²6020 inimmunocompetent BALB/c mice, we infected mice intravenously with 1×10⁶mc²6020 or 1×10⁶ of wild-type H37Rv. All mice infected with H37Rvsuccumbed to infection by 150 days, whereas mice infected with mc²6020survived for more than 400 days. After 3 weeks following intravenousinfection, no colonies of mc²6020 could be recovered from spleen, liveror lungs of infected mice. Interestingly, mice immunized subcutaneouslywith one or two doses of mc²6020 demonstrated protection against aerosolchallenge with virulent M. tuberculosis, which was comparable to theprotection afforded by BCG vaccination (Table 7). No pantothenate andlysine requiring auxotrophs were recovered from spleen or lungs of miceat 1, 2 or 3 months following subcutaneous immunization. Other studiesestablished that both mc²6020 and mc²6030 protects the a level ofprotection of mice against TB equivalent to the protection afforded byBCG (FIG. 19).

TABLE 7 Bacterial burden of virulent M. tuberculosis in uninoculatedmice and mice inoculated with BCG or one or two doses of mc²6020(ΔlysAΔpanCD) sc or one dose of mc²6020 iv. Experimental group Lung CFUs(log₁₀) Spleen CFUs (log₁₀) naive 6.03 ± 0.05^(a) 4.84 ± 0.27 BCG (1dose) sc 4.76 ± 0.19 *** 3.95 ± 0.18 * mc²6020 (1 dose) sc 5.05 ± 0.06*** 4.02 ± 0.11 * mc²6020 (2 doses) sc 5.09 ± 0.05 *** 4.06 ± 0.27mc²6020 (1 dose) iv 5.06 ± 0.11 *** 4.00 ± 0.15 * ^(a)Mean ± SEM p <0.001 = ***; p < 0.05 = *

These data clearly demonstrate the safety and immunogenicity of thesetwo double mutants of M. tuberculosis in mice.

The double deletion mutant mc²6030 (ΔRD1ΔpanCD) immunizes and protectsCD4^(−/−) mice from aerosolized M. tuberculosis challenge. We tested thehypothesis that the attenuated double deletion mutants could protectCD4-deficient mice, a model of HIV-infected humans, from aerosolized M.tuberculosis challenge. The results of these tests are summarized inFIG. 20. While 100% of the non-immunized CD4^(−/−) were dead by 38 days,100% of the mice immunized with either BCG or mc²6030 are alive at 120days post challenge. After 120 days, none of the immunized mice had anyoutward sign of disease. The double deletion mutants were also saferthan BCG in SCID mice, where all of the SCID mice died before 100 dayswhen inoculated with BCG, 100% and 25% of the mice survived inoculationwith mc²6020 and mc²6030, respectively (FIG. 21). This indicates thatimmunity against M. tuberculosis can be elicited in a CD4-independentmanner. These results also support the notion that effective antibody orCD8-mediated vaccines to malaria and HIV could be developed in thecontext of these attenuated M. tuberculosis strains.

EXAMPLE 7 Mycobacterum tuberculosis RD1 panCD is Safe and ProtectsCD4-Deficient Mice Against Tuberculosis in the Absence of CD8 Cells

Example Summary

Tuberculosis (TB) remains a leading cause of death due to an infectiousagent and is particularly devastating among HIV-infected individuals.The risk of disseminated BCG disease precludes the use of BCG vaccine inadults with known HIV infection or children with symptomatic AIDS. Thereis an urgent need for a safe and effective vaccine against tuberculosisfor immunocompetent, as well as immunodeficient individuals. Here, wereport that a mutant of Mycobacterium tuberculosis with two independentdeletions, in the RD1 region and panCD genes, is severely attenuated inimmunocompromised mice lacking T and B cells or mice lackinginterferon-gamma and significantly safer than the BCG vaccine. A singlesubcutaneous immunization with the ΔRD1 ΔpanCD mutant inducessignificant protective immune responses that prolong the survival ofimmunocompetent mice following a challenge with virulent M.tuberculosis. As a model that reflects the loss of CD4-cells associatedwith HIV infection, we tested whether M. tuberculosis ΔRD1 ΔpanCD couldimmunize and protect CD4-deficient mice against aerosol challenge withvirulent M. tuberculosis. Surprisingly, immunization with this mutantaffords significantly enhanced post-challenge survival to CD4-deficientmice than BCG vaccine. Furthermore, treatment of ΔRD1 ΔpanCD vaccinatedCD4^(−/−) mice with anti-CD8 antibody did not eliminate the protection,suggesting the role of a novel class of CD4⁻CD8⁻ cells in mediating thisprotection. Our results highlight the feasibility of generating multipledeletion mutants of M. tuberculosis that are non-revertible, highly safeand yet retain the ability to induce strong protective immunity againstTB in both immunocompetent and CD4-deficient mice.

Introduction

The global problem of tuberculosis (TB) is worsening, primarily as aresult of the growing HIV pandemic (Corbett et al., 2003). TB is aleading cause of death due an infectious agent, claiming more than 2million lives each year, with approximately 12% attributable to HIV. Theglobal TB problem is further worsened by the emergence of multi-drugresistant strains of Mycobacterium tuberculosis (Pablos-Mendez et al.,1998). Clearly, novel interventions in the form of an effective vaccineare urgently needed to reduce the disease burden of TB, particularly forHIV-infected individuals.

Vaccination with bacille Calmette-Guérin (BCG), a live attenuated strainof Mycobacterium bovis, induces protective immunity in children againstsevere and fatal forms of TB (Bloom and Fine, 1994; Rodrigues et al.,1993). However, the protection afforded by BCG vaccine against the mostprevalent pulmonary form of TB in adults is highly variable (0 to 80%)(Tuberculosis Prevention Trail, 1980; Fine, 1995). Although BCG has beenadministered to >3 billion people and has an overall excellent safetyrecord (Lotte et al., 1988), there have been several cases ofdisseminated BCG disease in individuals with mutations of their IL-12,or IL-12R genes following vaccination or infection with BCG (Altare etal., 1998; Casanova et al., 1995; de Jong et al., 1998). Likewise, therehave been numerous cases of disseminated BCG have been detected invaccinated children who subsequently developed AIDS (Talbot et al.,1997; von Reyn et al., 1987; Weltman and Rose, 1993; Braun and Cauthen,1992). Therefore, a safer and more effective vaccine than the currentlyused BCG vaccine is urgently needed to control TB in HIV-infectedindividuals.

The genetic basis for the primary attenuation of the widely used M.bovis derived BCG vaccine has been attributed to the loss ofapproximately 10 genes, named the region of difference, RD1 region(Lewis et al., 2003; Pym et al., 2002; Hsu et al., 2003). Comparativegenomic studies have revealed at least 129 open reading frames to bemissing from BCG strains in comparison to wild-type M. tuberculosis(Mahairas et al., 1996; Behr et al., 1999; Gordon et al., 1999). Thesemissing regions may encode potential antigenic determinants that couldincrease the immunogenicity of a vaccine, if it were derived from anattenuated strain of M. tuberculosis. Several live, attenuated M.tuberculosis vaccine candidates have been constructed by deleting genesrequired for growth in mice (Hondalus et al., 2000; Jackson et al.,1999; Smith et al., 2001) and have shown to confer some degree ofprotection against challenge infection with virulent M. tuberculosis.

We had previously reported the significant safety and immunogenicity ofa ΔpanCD mutant of M. tuberculosis in mice (Sambandamurthy et al.,2002). In an attempt to further enhance the safety of a M. tuberculosisderived vaccine, we deleted the panCD genes from the ΔRD1 mutant of M.tuberculosis that contains at least three attenuating mutations (Hsu etal., 2003). The resulting ΔRD1 ΔpanCD mutant has the safety features oftwo independent nonrevertible genomic deletions, which confersignificant attenuation even in immunodeficient mice that lack T and Bcells or mice that lack γ-interferon. In addition, the ΔRD1 ΔpanCDmutant undergoes limited replication in vivo; and thus conferssignificant long-term protection and survival in mice following a singledose vaccination. We also demonstrate that this attenuated TB vaccine issignificantly better than BCG in prolonging the survival ofCD4-deficient mice following an aerosol challenge with virulent M.tuberculosis and the protective immunity induced in theseimmunocompromised mice is largely mediated by a novel class of CD4⁻CD8⁻cells.

Materials and Methods

Media and Strains. M. tuberculosis H37Rv, M. tuberculosis Erdman and M.bovis BCG Pasteur were obtained from the Trudeau Culture Collection(Saranac Lake, N.Y.) and cultured as described earlier (Sambandamurthyet al., 2002). When required, pantothenate (24 μg/ml) or hygromycin (50μg/ml) was added. Stock strains were grown in Middlebrook 7H9 broth inroller bottles and harvested in mid-logarithmic growth phase, beforebeing stored in 1 ml vials at −70° C.

Construction of M. tuberculosis ΔRD1 ΔpanCD (mc26030) deletion mutant.Specialized transduction was employed to disrupt the chromosomal copy ofthe panCD genes from the unmarked M. tuberculosis ΔRD1 mutant (Hsu etal., 2003). The panCD and RD1 deletions were confirmed using PCR andSouthern blotting as described earlier (Hsu et al., 2003; Sambandamurthyet al., 2002).

Animal infections. C57BL/6, BALB/c, BALB/c SCID, C57BL/6 GKO (6-8 weeksold) were purchased from Jackson Laboratories and were infectedintravenously through the lateral tail vein. For time-to-death and mouseorgan CFU assays, BALB/c SCID mice were infected intravenously with 10²CFUs of M. tuberculosis H37Rv or 10⁵ CFUs of mc²6030. For GKO survivalexperiments, mice were infected intravenously with 10⁵ CFUs of M.tuberculosis H37Rv, BCG-P or the mc²6030 mutant. For mouse organ CFUassays, C57BL/6 mice were infected with 10⁶ CFUs of M. tuberculosisH37Rv or the mc²6030 mutant. At appropriate time points, groups of 4 or5 mice were sacrificed and the bacterial burden estimated as describedearlier (Sambandamurthy et al., 2002). The survival data and the CFUresults were statistically evaluated using either one-way analysis ofvariance or unpaired t-test analyses provided by the GraphPad InStatprogram. Pathologic examination was performed on tissues fixed in 10%buffered formalin. All animals were maintained in accordance withprotocols approved by the Albert Einstein College of Medicine and theCenter for Biologics Evaluation and Research Institutional Animal Careand Use Committee.

Vaccination Studies. Groups of C57BL/6 mice (5 mice per group) werevaccinated subcutaneously, either once or twice (6 weeks apart), with10⁶ CFUs of mc²6030. Control mice were vaccinated subcutaneously with10⁶ CFUs of M. bovis BCG-Pasteur. At either 3 or 8 months after theinitial immunization with mc²6030 or BCG, the mice were aerogenicallychallenged with approximately 100-200 CFUs of M. tuberculosis Erdmanstrain. At 28 days following aerosol challenge, the challenged mice weresacrificed and the bacterial numbers in the lung and spleen wereenumerated as described earlier (Sambandamurthy et al., 2002).

For immunization studies in CD4^(−/−) mice (Jackson Laboratories), micewere vaccinated subcutaneously with a single dose of 10⁶ CFUs of mc²6030or BCG-P and challenged three months later through the aerosol route.For the survival studies, 5 to 6 mice per group were maintained untilthey became moribund and had to be euthanized. Five mice were sacrificedafter 24 hours to confirm the size of the challenge dose following theaerosol challenge.

For the CD8 depletion studies, CD4⁴ mice were immunized with a singlesubcutaneous dose of mc²6030 and then treated intraperitoneally withanti-CD8 monoclonal antibody (clone 243, Harlan Bioproducts,Indianapolis Ind.) at two days before, the day of the tuberculouschallenge, and then twice per week (0.2 mg/ml per dose) until the micewere sacrificed. The effectiveness of the anti-CD8 antibody treatmentwas confirmed by flow cytometry after staining peripheral blood and lunglymphocytes with Cy-Chrome-conjugated rat anti-mouse CD8a (ly-2)monoclonal antibody (Pharmingen, San Diego, Calif.).

To assess the role of IFN-γ in mediating protection, GKO mice (n=10)were vaccinated with 10⁶ CFU of mc²6030 or BCG-P and challenged threemonths later through the aerosol route and followed for survival.

Results

mc²6030 is highly attenuated in immunodeficient mice. In an attempt todevelop a safe and effective vaccine strain, the panCD genes(Sambandamurthy et al., 2002) were deleted from an unmarked ΔRD1 mutantof M. tuberculosis using specialized transduction (Bardarov et al.,2002). The deletions in the RD1 region and panCD genes were confirmedusing PCR and Southern blotting (data not shown). Strain mc²6030 isauxotrophic for pantothenate; no revertants were recovered when 10¹¹ CFUof mc²6030 strain were plated on minimal media, demonstrating themutations to be highly stable and non-revertible.

An important pre-requisite for any live attenuated vaccine is their safeuse even in immunodeficient hosts. To assess the attenuation of thismutant, severe combined immunodeficient (SCID) mice, a highly stringentmodel for safety, were infected intravenously with H37Rv or mc²6030.Mice infected with 10² CFU of M. tuberculosis H37Rv strain died within 4weeks post-infection. Interestingly, 60% of SCID mice infected with 10⁵CFU of mc²6030 survived for over 350 days (FIG. 22A). Mice infected withH37Rv showed a rapid increase in bacterial numbers in the lungs, spleenand liver by 3 weeks. In contrast, the bacterial numbers in the spleenof mc²6030-infected mice remained relatively constant throughout thecourse of infection (FIG. 22B). Bacterial numbers in the lungs ofmc²6030-infected SCID mice showed a decrease in the first 3 weeks ofinfection, but gradually increased to reach 10⁸ CFUs by 350 days (FIG.22C). The bacterial titers were constant in the liver throughout thecourse of infection except for a sharp decline at 3 weeks (data notshown).

Overwhelming evidence from humans and animal models has implicated IFN-γto be a key cytokine in the control of M. tuberculosis infection (Flynnand Chan, 2001). IFN-γ knockout (GKO) mice are extremely sensitive totuberculous infection (Cooper et al., 1993; Flynn et al., 1993) andindividuals defective in genes for IFN-γ receptors have increasedsusceptibility to disseminated mycobacterial disease (Ottenhoff et al.,1998). As an additional assessment of the safety of this attenuatedmutant, GKO mice were infected intravenously with 10⁵ CFU of M.tuberculosis H37Rv, BCG Pasteur (BCG-P) or mc²6030. All of the GKO miceinfected with H37Rv (mean survival time, MST, 21 days) or BCG-P (MST, 93days) succumbed to the tuberculous infection within 4 months,whereas >60% of mice infected with mc²6030 were alive at 335 days (FIG.22 d). The extreme susceptibility of immunocompromised mice to M.tuberculosis and BCG infections is consistent with data from previousstudies using SCID (Sambandamurthy et al., 2002; Weber et al., 2000) andGKO mice (Dalton et al., 1993). Overall, our data indicate that mc²6030is significantly more attenuated and less virulent in SCID and GKO micethan the widely used BCG vaccine strain.

mc²6030 undergoes limited replication in mice. To evaluate the effect ofthe multiple mutations in strain mc²6030 on bacterial growth in vivo,immunocompetent BALB/c or C57BL/6 mice were infected intravenously. AllBALB/c mice infected with 10⁵ CFU of H37Rv succumbed to the resultinginfection by 168 days (MST=134 days). In contrast, all mice infectedwith 10⁵ CFU of mc²6030 survived for over 400 days (data not shown).Similarly, C57BL/6 mice infected with 10⁶ CFU of H37Rv succumbed toinfection by 260 days (MST=196 days). All mice infected with 10⁶ CFUs ofmc²6030 survived over 400 days post-infection (FIG. 22E).Bacteriological culture results generally showed a decline in thenumbers of H37Rv and mc²6030 organisms in the spleen (FIG. 22F) andliver (data not shown) of C57BL/6 mice after infection. Interestingly,the pulmonary bacterial CFU numbers for both the virulent H37Rv strainand the attenuated mutant reached a constant level at 3 monthspost-infection (FIG. 22G). Importantly, the H37Rv CFU values were atleast 100-fold higher than the mc²6030 CFUs in all organs tested at 200days after the infection.

Histopathological examination of organs from infected mice confirmed themarked attenuation of the deletion mutant. At 3 weeks post-infection, anintravenous injection of mc²6030 (FIG. 22H) had caused only rare, mildperivascular, lymphocytic infiltrates. The spleens were slightlyenlarged with mononuclear cell infiltration in the red pulp. Also,multifocal, mild infiltrations of macrophages and neutrophils were seenin the liver and no acid-fast bacilli were detected. This contrastedwith the severe pneumonia (FIG. 22I), markedly enlarged spleens, severediffuse granulomatous hepatitis, and the overwhelming bacterial burdenseen in mice infected with the H37Rv strain.

mc²6030 induces short and long-term protection in immunocompetent mice.Having assessed the safety and growth kinetics of mc²6030 in bothimmunodeficient and immunocompetent mice, we evaluated the protectiveimmune responses induced by this attenuated strain. As a test of itsvaccine potential, C57BL/6 mice were immunized subcutaneously with 10⁶CFUs of mc²6030 and then were challenged 3 months later with a low doseof virulent M. tuberculosis Erdman by the aerosol route. Followingsubcutaneous immunization, the immunizing mc²6030 mutant bacteria couldnot be cultured from the spleens or lungs of mice at 8 and 12 weekspostvaccination. At 28 days post-aerosol challenge, mice that wereimmunized 3 months earlier with a single dose of mc²6030 showed asignificant reduction in the lung (P<0.01) and spleen (P<0.01) bacterialCFU values as compared to naïve mice. Consistent with published results,mice vaccinated with 10⁶ CFUs of BCG showed similar CFU reductions inthe lungs (P<0.001) and spleen (P<0.001) as compared to the naïvecontrols (FIG. 23A,B).

In order to assess the duration and persistence of the memory immuneresponse, vaccinated and control mice were challenged through theaerosol route 8 months after a single dose vaccination. In the naïvecontrols at four weeks post-challenge, the bacterial numbers increaseddramatically in the lung and substantial dissemination and growth in thespleen were also observed. Strikingly, mice vaccinated with a singledose of mc²6030 or BCG-P displayed statistically significant reductionin bacterial numbers in the lungs (P<0.001) and spleen (P<0.001)relative to naïve controls (FIG. 23C D). Our data clearly demonstratethat a single dose of mc²6030 induces a potent and long-lastingprotective immune response that is effective in controlling a virulentM. tuberculosis challenge in the lungs and spleen of mice even after 8months following the primary immunization.

Another relevant measure of vaccine effectiveness is the relativesurvival periods for immunized mice following a challenge with virulentorganisms (33). To evaluate whether vaccination with mc²6030 increasedmean survival times, C57BL/6 mice that had been immunized subcutaneouslywith a single dose of mc²6030 (or BCG-P) were challenged 3 months laterwith virulent M. tuberculosis Erdman by the aerosol route and followedfor survival. All of the unvaccinated mice succumbed to the tuberculousinfection (MST=95±22 days) following the aerosol challenge. In contrast,the survival periods of mice vaccinated with either mc²6030 (MST, 267±14days) or BCG-P (MST, 268±16 days) were significantly extended (P<0.001)relative to naïve mice (FIG. 23 e).

mc²6030 protects CD4-deficient mice significantly better than BCGagainst anaerosolized TB challenge. Tuberculosis remains the largestattributable cause of death in HIV-infected individuals (Whalen et al.,2000). HIV infection leads to a loss of CD4⁺T cells and previous studieshave demonstrated that mice deficient in CD4⁺T cells are highlysusceptible to M. bovis BCG (Ladel et al., 1995) and M. tuberculosisinfection (Mogues et al., 2001; Caruso et al., 1999). Since BCGvaccination is contraindicated in HIV-infected individuals, we wanted totest if the more attenuated strain, mc²6030, could protect CD4-deficientmice from experimental tuberculosis. CD4-deficient mice were vaccinatedsubcutaneously with a single dose of 10⁶ CFUs of either mc²6030 or BCG-Pand then aerogenically challenged with 100-200 CFUs of M. tuberculosisErdman three months later. At 28 days post-aerosol challenge, thebacterial burden in the lungs (P<0.001) and spleen (P<0.001) of themc²6030 and BCG-P vaccinated mice was decreased by greater than 99% (>2log CFU) relative to naïve controls (FIG. 24A,B).

To further evaluate the long-lived protection induced by mc²6030,CD4^(−/−) mice were vaccinated, challenged 3 months later with virulentM. tuberculosis Erdman through the aerosol route, and followed forsurvival. All of the naïve mice died within 29 days (MST=27±2 days) ofthe low dose tuberculous aerogenic challenge (FIG. 24C). Strikingly, themean survival time for the CD4^(−/−) mice vaccinated with a single doseof mc²6030 was 214±18 days, a nearly eight-fold extension of thesurvival period compared to naïves. In contrast, the BCG vaccinated micesurvived 158±23 days. The 56-day extension of the MST for themc²6030-immunized mice relative to BCG vaccinated animals represented asignificantly improved outcome (P<0.05) for CD4^(−/−) mice immunizedwith the M. tuberculosis mutant strain.

Histologically, significant differences were observed between thevaccinated groups and naïve CD4^(−/−) controls. The unvaccinated micedeveloped severe lung lesions with multiple large inflammatory nodules;some coalescing and spreading to areas of extensive diffuse pneumonia.The inflammatory response consisted of macrophages, numerousneutrophils, accompanied by low numbers of lymphocytes. There were largenumbers of acid-fast organisms in the lesions (FIG. 25A,D). In contrast,mice vaccinated with mc²6030 (FIGS. 25B,E) or BCG (FIG. 25C,F) showedreduced severity of the lung lesions. These mice showed scattereddistinct lesions, adjacent to airways and localized, which remainedsmaller in diameter than in unvaccinated mice. These multifocal areaswere composed of macrophages and numerous lymphocytes. There was amarked reduction in the numbers of acid-fast organisms compared to naïvecontrols.

The ability of mc²6030-vaccinated CD4^(−/−) mice to control thetuberculous challenge suggests the potential role of CD8⁺T cells inmediating this protection. In order to directly demonstrate the role ofCD8⁺T cells in this protective response, CD4^(−/−) mice that werevaccinated with mc²6030 were treated with anti-CD8 monoclonal antibodyand subsequently challenged with virulent M. tuberculosis through theaerosol route. Flow cytometric analysis showed that the anti-CD8antibody treatment had depleted >99% of CD8⁺T cells from the lungs andperipheral blood. Surprisingly, repeated injections of the anti-CD8antibody did not reduce the vaccine-mediated protective immune response.At 28 days post-aerosol challenge, the bacterial burdens in the lungsand spleens of the anti-CD8 antibody-treated immunized CD4^(−/−) miceand nontreated vaccinated mice were similar; significant reductions inthe pulmonary and splenic bacterial CFUs, relative to nonimmunizedcontrols (>2 log₁₀ in the lungs and >1 log₁₀ in the spleen) weredetected for each vaccine group (FIG. 24D,E).

To examine the role of IFN-γ in mediating the anti-tuberculous immunityevoked by the mutant M. tuberculosis strain, GKO mice were vaccinatedwith mc²6030 or BCG-P and challenged with M. tuberculosis. Immunizationof GKO mice with mc²6030 or BCG-P did not significantly increase thesurvival period in comparison to unvaccinated controls with all mice ineach of the three groups succumbing to the resulting infection within 30days. Interestingly, 4 out of 10 BCG-vaccinated GKO mice died ofdisseminated BCG infection even before the aerosol challenge (FIG. 24F).

Discussion

Vaccination remains the most cost-effective and proven strategy toprotect mankind against infectious agents (Bloom, 1989). The eradicationof smallpox and the near elimination of poliomyelitis demonstrate thepotential of one class of vaccines, the live attenuated vaccines.However, a major obstacle to the development of these vaccines is thedifficulty in achieving a satisfactory level of attenuation withoutseverely compromising the immunogenicity of the vaccine strain. Recentadvances in the molecular biology of mycobacteria have been used toconstruct several attenuated mutants of M. tuberculosis as candidatesfor a vaccine (Hondalus et al., 2000; Jackson et al., 1999; Smith etal., 2001). An M. tuberculosis-derived vaccine may be more efficaciousand induce a qualitatively better protective immune response than an M.bovis-derived BCG vaccine because the immunogenicity of BCG seem to havedeclined following extensive passage over the years (Behr and Small,1997) and M. tuberculosis mutants should express an antigenic profilethat is nearly identical to virulent TB.

The deletion of the RD1 region (the primary attenuating deletion in allBCG vaccine strains) from M. tuberculosis leads to attenuation of theresulting mutant strain (Lewis et al., 2003; Hsu et al., 2003). However,that strain is relatively more virulent than the currently used BCGvaccine in immunodeficient SCID mice. In contrast, a multiple deletionmutant of M. tuberculosis (mc²6030) harboring deletions in the RD1region and the panCD genes is severely attenuated in SCID mice. Thismutant is considerably less virulent than the single ΔRD1 or ΔpanCD M.tuberculosis mutants or BCG in this mouse model. Interestingly, our dataalso show that the mc²6030 mutant is markedly more attenuated than thewidely used BCG vaccine in mice lacking IFN-γ.

While live strains must be attenuated, they should retain their capacityto undergo limited in vivo replication (McKenney et al., 1999; Kanai andYanagisawa, 1955; Brant et al., 2002). The failure of earlier auxotrophsof M. tuberculosis to induce a potent protective immune response couldbe due to insufficient in vivo replication. The capacity of mc²6030mutant to undergo limited replication without causing detrimentalpathology in the tissues of infected wild-type mice likely contributesto its ability to generate long-term immunity in mice. The hallmark ofimmunological memory and vaccination is the ability to mount anaccelerated response to reinfection, which is achieved by the generationof long-lived memory T cells. The induction and long-term maintenance ofcellular immune responses is the primary goal of a vaccine for anintracellular pathogen such as Mycobacterium tuberculosis (Seder andHill, 2000). For the mc²6030 strain, the protective response seen at 8months following a single immunization reflects the generation oflong-term memory response. An additional proof of this induction ofimmunological memory was demonstrated in the survival studies; micevaccinated with mc²6030 survived significantly longer than control micefollowing a tuberculous challenge.

HIV-infection is a major risk factor for both primary and reactivationaldisease with M. tuberculosis in humans. The annual risk of developing TBin individuals coinfected with TB and HIV is 10%, while immunocompetentindividuals have only a 10% lifetime risk of developing diseasefollowing infection. Therefore, HIV-1 infected individuals remain acritical cohort of patients suitable for vaccination. However, candidatevaccines may not be sufficiently immunogenic to stimulate protectiveimmunity in potentially immunodeficient HIV-1 infected patients withimpaired or defective CD4⁺ T cell responses. To test this principle, weexplored whether immunedeficient mice lacking CD4⁺ T cells could beprotected against TB by vaccination with a safe mutant of M.tuberculosis. Interestingly, our study clearly demonstrates the abilityof a single dose vaccination with mc²6030 to confer significantprotection in CD4^(−/−) mice following an aerosol challenge withvirulent M. tuberculosis. Reduced organ bacterial burdens, prolongedsurvival and a milder pulmonary histopathology were seen in vaccinatedmice relative to naïve mice lacking CD4⁺ T cells. Importantly, themc²6030 vaccinated CD4^(−/−) mice survived significantly longer (56days) post-challenge than BCG-vaccinated animals. The immunogenicty ofthis mutant and its reduced virulence likely contribute to the improvedoutcome in these immunocompromised mice. Overall, these data stronglysuggest that further testing of this attenuated M. tuberculosis strainin other immunocompromised models is warranted with a goal of developingan effective TB vaccine in HIV-1 infected individuals.

The biological basis for the enhanced effectiveness of the mc²6030strain in mice lacking CD4⁺ T cells has not been completely elucidated.Clearly, the specific immune mechanisms associated with protection aftervaccination and challenge in CD4⁺ deficient mice are of substantialinterest because several studies have suggested that CD4⁺ T cells areabsolutely required to generate anti-tuberculosis protective immunity(Flynn and Chan, 2001). Since CD8⁺ T cells have been shown to play animportant role in the control of acute tuberculosis (Behar et al., 1999;Sousa et al., 2000; Serbina and Flynn, 2001) and persistent infection(van Pinxteren et al., 2000b), we hypothesized that CD8 cells mediatedthe protective immunity generated by vaccination of CD4^(−/−) mice withthe mc²6030 strain. Although several studies have demonstrated that CD4⁺T cell help is important in the activation and differentiation of naïveCD8⁺ T cells into cytotoxic effector cells (Cardin et al., 1996; vanHerrath et al., 1996; Wild et al., 1999), recent evidence suggests thatvaccine immunity can be generated in the absence of CD4⁺ T cells (Bulleret al., 1987; Stevenson et al., 1998; Wuthrich et al., 2003) and CD8⁺ Tcells can provide self-help when they are present at a sufficiently highprecursor frequency (Wang et al., 2001; Mintern et al., 2002′).Consistent with this premise, potent CD8 mediated anti-mycobacterialprotective immunity was generated following immunization with a DNAvaccine cocktail in CD4-deficient mice (Steven Derrick and SheldonMorris, Infection and Immunity, in press). Surprisingly, treatment ofvaccinated mice with an anti-CD8 antibody did not reduce the protectiveimmune response in CD4^(−/−) mice induced by immunization with the M.tuberculosis mutant. This result suggests that CD4⁻CD8⁻ cells maycontribute to the anti-tuberculous protection. These anti-CD8 antibodydata are consistent with the previously described role ofdouble-negative T cells in protecting against infections withintracellular pathogens. In a Listeria model, Dunn and North showed theresolution of primary listeriosis and acquired resistance to secondaryinfection to be predominantly mediated by double-negative T cells (Dunnand North, 1991). More recently, Cowley and Elkins reported thatsecondary immunity to the Francisella tularensis live vaccine strain waspartially mediated by a unique CD4⁻CD8⁻ T cell population (Cowley andElkins, 2003). Further studies are currently in progress to directlydetermine whether double-negative T cells are mediating the ΔRD1 ΔpanCDvaccine strain-induced protective immunity in CD4-deficient mice andwhether CD4⁻CD8⁻ T cells are generally involved in the protectiveresponses elicited by live attenuated strains against intracellularpathogens.

Overall, our results demonstrate that it is possible to generate twoindependent unlinked deletions, each containing multiple attenuatingmutations into M. tuberculosis to generate a safe mutant that can remainimmunogenic and provide protective immunity against airborne infectionwith virulent M. tuberculosis in mice. The protection and safety datafrom immunocompromised and immunocompetent mice and the lack ofreversion make this multiple deletion mutant a viable vaccine candidatefor humans. The numerous advantages of BCG, i.e., affordability, safety,ability to be used in newborns, and its use as a recombinant vaccinedelivery vector should be also applicable to the highly attenuatedmc²6030 candidate. Since the mc²6030 strain is more attenuated than BCGand yet is more protective in immunocompromised mice, the evaluation ofthe mc²6030 mutant as a vaccine in other animal models of tuberculosis,including nonhuman primate models, is clearly warranted.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

SEQ ID NO:s

SEQ ID NO:1-An RD1 region of Mycobacterium tuberculosis H37Rv. Bases4350263-4359716 of the genome of M. tuberculosis H37Rv, as provided in GenBank Accession No. NC000962. 4350263gatcgtgg gtgccgccgg ggggatgccg ccgatggcac 4350301cgctggcccc gttattgccg gcggcggcag atatcgggtt gcacatcatt gtcacctgtc4350361agatgagcca ggcttacaag gcaaccatgg acaagttcgt cggcgccgca ttcgggtcgg4350421gcgctccgac aatgttcctt tcgggcgaga agcaggaatt cccatccagt gagttcaagg4350481tcaagcggcg cccccctggc caggcatttc tcgtctcgcc agacggcaaa gaggtcatcc4350541aggcccccta catcgagcct ccagaagaag tgttcgcagc acccccaagc gccggttaag4350601attatttcat tgccggtgta gcaggacccg agctcagccc ggtaatcgag ttcgggcaat4350661gctgaccatc gggtttgttt ccggctataa ccgaacggtt tgtgtacggg atacaaatac4350721agggagggaa gaagtaggca aatggaaaaa atgtcacatg atccgatcgc tgccgacatt4350781ggcacgcaag tgagcgacaa cgctctgcac ggcgtgacgg ccggctcgac ggcgctgacg4350841tcggtgaccg ggctggttcc cgcgggggcc gatgaggtct ccgcccaagc ggcgacggcg4350901ttcacatcgg agggcatcca attgctggct tccaatgcat cggcccaaga ccagctccac4350961cgtgcgggcg aagcggtcca ggacgtcgcc cgcacctatt cgcaaatcga cgacggcgcc4351021gccggcgtct tcgccgaata ggcccccaac acatcggagg gagtgatcac catgctgtgg4351081cacgcaatgc caccggagct aaataccgca cggctgatgg ccggcgcggg tccggctcca4351141atgcttgcgg cggccgcggg atggcagacg ctttcggcgg ctctggacgc tcaggccgtc4351201gagttgaccg cgcgcctgaa ctctctggga gaagcctgga ctggaggtgg cagcgacaag4351261gcgcttgcgg ctgcaacgcc gatggtggtc tggctacaaa ccgcgtcaac acaggccaag4351321acccgtgcga tgcaggcgac ggcgcaagcc gcggcataca cccaggccat ggccacgacg4351381ccgtcgctgc cggagatcgc cgccaaccac atcacccagg ccgtccttac ggccaccaac4351441ttcttcggta tcaacacgat cccgatcgcg ttgaccgaga tggattattt catccgtatg4351501tggaaccagg cagccctggc aatggaggtc taccaggccg agaccgcggt taacacgctt4351561ttcgagaagc tcgagccgat ggcgtcgatc cttgatcccg gcgcgagcca gagcacgacg4351621aacccgatct tcggaatgcc ctcccctggc agctcaacac cggttggcca gttgccgccg4351681gcggctaccc agaccctcgg ccaactgggt gagatgagcg gcccgatgca gcagctgacc4351741cagccgctgc agcaggtgac gtcgttgttc agccaggtgg gcggcaccgg cggcggcaac4351801ccagccgacg aggaagccgc gcagatgggc ctgctcggca ccagtccgct gtcgaaccat4351861ccgctggctg gtggatcagg ccccagcgcg ggcgcgggcc tgctgcgcgc ggagtcgcta4351921cctggcgcag gtgggtcgtt gacccgcacg ccgctgatgt ctcagctgat cgaaaagccg4351981gttgccccct cggtgatgcc ggcggctgct gccggatcgt cggcgacggg tggcgccgct4352041ccggtgggtg cgggagcgat gggccagggt gcgcaatccg gcggctccac caggccgggt4352101ctggtcgcgc cggcaccgct cgcgcaggag cgtgaagaag acgacgagga cgactgggac4352161gaagaggacg actggtgagc tcccgtaatg acaacagact tcccggccac ccgggccgga4352221agacttgcca acattttggc gaggaaggta aagagagaaa gtagtccagc atggcagaga4352281tgaagaccga tgccgctacc ctcgcgcagg aggcaggtaa tttcgagcgg atctccggcg4352341acctgaaaac ccagatcgac caggtggagt cgacggcagg ttcgttgcag ggccagtggc  4352401gcggcgcggc ggggacggcc gcccaggccg cggtggtgcg cttccaagaa gcagccaata4352461agcagaagca ggaactcgac gagatctcga cgaatattcg tcaggccggc gtccaatact4352521cgagggccga cgaggagcag cagcaggcgc tgtcctcgca aatgggcttc tgacccgcta4352581atacgaaaag aaacggagca aaaacatgac agagcagcag tggaatttcg cgggtatcga4352641ggccgcggca agcgcaatcc agggaaatgt cacgtccatt cattccctcc ttgacgaggg4352701gaagcagtcc ctgaccaagc tcgcagcggc ctggggcggt agcggttcgg aggcgtacca4352761gggtgtccag caaaaatggg acgccacggc taccgagctg aacaacgcgc tgcagaacct4352821ggcgcggacg atcagcgaag ccggtcaggc aatggcttcg accgaaggca acgtcactgg4352881gatgttcgca tagggcaacg ccgagttcgc gtagaatagc gaaacacggg atcgggcgag4352941ttcgaccttc cgtcggtctc gccctttctc gtgtttatac gtttgagcgc actctgagag4353001gttgtcatgg cggccgacta cgacaagctc ttccggccgc acgaaggtat ggaagctccg4353061gacgatatgg cagcgcagcc gttcttcgac cccagtgctt cgtttccgcc ggcgcccgca4353121tcggcaaacc taccgaagcc caacggccag actccgcccc cgacgtccga cgacctgtcg4353181gagcggttcg tgtcggcccc gccgccgcca cccccacccc cacctccgcc tccgccaact4353241ccgatgccga tcgccgcagg agagccgccc tcgccggaac cggccgcatc taaaccaccc4353301acacccccca tgcccatcgc cggacccgaa ccggccccac ccaaaccacc cacacccccc4353361atgcccatcg ccggacccga accggcccca cccaaaccac ccacacctcc gatgcccatc4353421gccggacctg cacccacccc aaccgaatcc cagttggcgc cccccagacc accgacacca4353481caaacgccaa ccggagcgcc gcagcaaccg gaatcaccgg cgccccacgt accctcgcac4353541gggccacatc aaccccggcg caccgcacca gcaccgccct gggcaaagat gccaatcggc4353601gaacccccgc ccgctccgtc cagaccgtct gcgtccccgg ccgaaccacc gacccggcct4353661gccccccaac actcccgacg tgcgcgccgg ggtcaccgct atcgcacaga caccgaacga4353721aacgtcggga aggtagcaac tggtccatcc atccaggcgc ggctgcgggc agaggaagca4353781tccggcgcgc agctcgcccc cggaacggag ccctcgccag cgccgttggg ccaaccgaga4353841tcgtatctgg ctccgcccac ccgccccgcg ccgacagaac ctccccccag cccctcgccg4353901cagcgcaact ccggtcggcg tgccgagcga cgcgtccacc ccgatttagc cgcccaacat4353961gccgcggcgc aacctgattc aattacggcc gcaaccactg gcggtcgtcg ccgcaagcgt4354021gcagcgccgg atctcgacgc gacacagaaa tccttaaggc cggcggccaa ggggccgaag4354081gtgaagaagg tgaagcccca gaaaccgaag gccacgaagc cgcccaaagt ggtgtcgcag4354141cgcggctggc gacattgggt gcatgcgttg acgcgaatca acctgggcct gtcacccgac4354201gagaagtacg agctggacct gcacgctcga gtccgccgca atccccgcgg gtcgtatcag4354261atcgccgtcg tcggtctcaa aggtggggct ggcaaaacca cgctgacagc agcgttgggg4354321tcgacgttgg ctcaggtgcg ggccgaccgg atcctggctc tagacgcgga tccaggcgcc4354381ggaaacctcg ccgatcgggt agggcgacaa tcgggcgcga ccatcgctga tgtgcttgca4354441gaaaaagagc tgtcgcacta caacgacatc cgcgcacaca ctagcgtcaa tgcggtcaat4354501ctggaagtgc tgccggcacc ggaatacagc tcggcgcagc gcgcgctcag cgacgccgac4354561tggcatttca tcgccgatcc tgcgtcgagg ttttacaacc tcgtcttggc tgattgtggg4354621gccggcttct tcgacccgct gacccgcggc gtgctgtcca cggtgtccgg tgtcgtggtc4354681gtggcaagtg tctcaatcga cggcgcacaa caggcgtcgg tcgcgttgga ctggttgcgc4354741aacaacggtt accaagattt ggcgagccgc gcatgcgtgg tcatcaatca catcatgccg4354801ggagaaccca atgtcgcagt taaagacctg gtgcggcatt tcgaacagca agttcaaccc4354861ggccgggtcg tggtcatgcc gtgggacagg cacattgcgg ccggaaccga gatttcactc4354921gacttgctcg accctatcta caagcgcaag gtcctcgaat tggccgcagc gctatccgac4354981gatttcgaga gggctggacg tcgttgagcg cacctgctgt tgctgctggt cctaccgccg4355041cgggggcaac cgctgcgcgg cctgccacca cccgggtgac gatcctgacc ggcagacgga4355101tgaccgattt ggtactgcca gcggcggtgc cgatggaaac ttatattgac gacaccgtcg4355161cggtgctttc cgaggtgttg gaagacacgc cggctgatgt actcggcggc ttcgacttta4355221ccgcgcaagg cgtgtgggcg ttcgctcgtc ccggatcgcc gccgctgaag ctcgaccagt4355281cactcgatga cgccggggtg gtcgacgggt cactgctgac tctggtgtca gtcagtcgca4355341ccgagcgcta ccgaccgttg gtcgaggatg tcatcgacgc gatcgccgtg cttgacgagt4355401cacctgagtt cgaccgcacg gcattgaatc gctttgtggg ggcggcgatc ccgcttttga4355461ccgcgcccgt catcgggatg gcgatgcggg cgtggtggga aactgggcgt agcttgtggt4355521ggccgttggc gattggcatc ctggggatcg ctgtgctggt aggcagcttc gtcgcgaaca4355581ggttctacca gagcggccac ctggccgagt gcctactggt cacgacgtat ctgctgatcg4355641caaccgccgc agcgctggcc gtgccgttgc cgcgcggggt caactcgttg ggggcgccac4355701aagttgccgg cgccgctacg gccgtgctgt ttttgacctt gatgacgcgg ggcggccctc4355761ggaagcgtca tgagttggcg tcgtttgccg tgatcaccgc tatcgcggtc atcgcggccg4355821ccgctgcctt cggctatgga taccaggact gggtccccgc gggggggatc gcattcgggc4355881tgttcattgt gacgaatgcg gccaagctga ccgtcgcggt cgcgcggatc gcgctgccgc4355941cgattccggt acccggcgaa accgtggaca acgaggagtt gctcgatccc gtcgcgaccc4356001cggaggctac cagcgaagaa accccgacct ggcaggccat catcgcgtcg gtgcccgcgt4356061ccgcggtccg gctcaccgag cgcagcaaac tggccaagca acttctgatc ggatacgtca4356121cgtcgggcac cctgattctg gctgccggtg ccatcgcggt cgtggtgcgc gggcacttct4356181ttgtacacag cctggtggtc gcgggtttga tcacgaccgt ctgcggattt cgctcgcggc4356241tttacgccga gcgctggtgt gcgtgggcgt tgctggcggc gacggtcgcg attccgacgg4356301gtctgacggc caaactcatc atctggtacc cgcactatgc ctggctgttg ttgagcgtct4356361acctcacggt agccctggtt gcgctcgtgg tggtcgggtc gatggctcac gtccggcgcg4356421tttcaccggt cgtaaaacga actctggaat tgatcgacgg cgccatgatc gctgccatca4356481ttcccatgct gctgtggatc accggggtgt acgacacggt ccgcaatatc cggttctgag4356541ccggatcggc tgattggcgg ttcctgacag aacatcgagg acacggcgca ggtttgcata4356601ccttcggcgc ccgacaaatt gctgcgattg agcgtgtggc gcgtccggta aaatttgctc4356661gatggggaac acgtatagga gatccggcaa tggctgaacc gttggccgtc gatcccaccg4356721gcttgagcgc agcggccgcg aaattggccg gcctcgtttt tccgcagcct ccggcgccga4356781tcgcggtcag cggaacggat tcggtggtag cagcaatcaa cgagaccatg ccaagcatcg4356841aatcgctggt cagtgacggg ctgcccggcg tgaaagccgc cctgactcga acagcatcca4356901acatgaacgc ggcggcggac gtctatgcga agaccgatca gtcactggga accagtttga4356961gccagtatgc attcggctcg tcgggcgaag gcctggctgg cgtcgcctcg gtcggtggtc4357021agccaagtca ggctacccag ctgctgagca cacccgtgtc acaggtcacg acccagctcg4357081gcgagacggc cgctgagctg gcaccccgtg ttgttgcgac ggtgccgcaa ctcgttcagc4357141tggctccgca cgccgttcag atgtcgcaaa acgcatcccc catcgctcag acgatcagtc4357201aaaccgccca acaggccgcc cagagcgcgc agggcggcag cggcccaatg cccgcacagc4357261ttgccagcgc tgaaaaaccg gccaccgagc aagcggagcc ggtccacgaa gtgacaaacg4357321acgatcaggg cgaccagggc gacgtgcagc cggccgaggt cgttgccgcg gcacgtgacg4357381aaggcgccgg cgcatcaccg ggccagcagc ccggcggggg cgttcccgcg caagccatgg4357441ataccggagc cggtgcccgc ccagcggcga gtccgctggc ggcccccgtc gatccgtcga4357501ctccggcacc ctcaacaacc acaacgttgt agaccgggcc tgccagcggc tccgtctcgc4357561acgcagcgcc tgttgctgtc ctggcctcgt cagcatgcgg cggccagggc ccggtcgagc4357621aacccggtga cgtattgcca gtacagccag tccgcgacgg ccacacgctg gacggccgcg4357681tcagtcgcag tgtgcgcttg gtgcagggca atctcctgtg agtgggcagc gtaggcccgg4357741aacgcccgca gatgagcggc ctcgcggccg gtagcggtgc tggtcatggg cttcatcagc4357801tcgaaccaca gcatgtgccg ctcatcgccc ggtggattga catccaccgg cgccggcggc4357861aacaagtcga gcaaacgctg atcggtagtg tcggccagct gagccgccgc cgaggggtcg4357921acgacctcca gccgcgaccg gcccgtcatt ttgccgctct ccggaatgtc atctggctcc4357981agcacaatct tggccacacc gggatccgaa ctggccaact gctccgcggt accgatcacc4358041gcccgcagcg tcatgtcgtg gaaagccgcc caggcttgca cggccaaaac cgggtaggtg4358101gcacagcgtg caatttcgtc aaccgggatt gcgtgatccg cgctggccaa gtacacctta4358161ttcggcaatt ccatcccgtc gggtatgtag gccagcccat agctgttggc cacgacgatg4358221gaaccgtcgg tggtcaccgc ggtgatccag aagaacccgt agtcgcccgc gttgttgtcg4358281gacgcgttga gcgccgccgc gatgcgtcgc gccaaccgca gcgcatcacc gcggccacgc4358341tggcgggcgc tggcagctgc agtggcggcg tcgcgtgccg cccgagccgc cgacaccggg4358401atcatcgaca ccggcgtacc gtcatctgca gactcgctgc gatcgggttt gtcgatgtga4358461tcggtcgacg gcgggcgggc aggaggtgcc gtccgcgccg aggccgcccg cgtgctcggt4358521gccgccgcct tgtccgaggt agccaccggc gcccgcccag tggcagcatg cgaccccgcg4358581cccgaggccg cggccgtacc cacgctcgaa cgcgcgcccg ctcccacggc ggtaccgctc4358641ggcgcggcgg ccgccgcccg tgcgcccggg acaccggacg ccgcagccgg cgtcaccgac4358701gcggcggatt cgtccgcatg ggcaggcccc gactgcgtcc ccccgcccgc atgctggccc4358761ggcacaccag gttgctccgc caacgccgcg ggtttgacgt gcggcgccgg ctcgccccct4358821ggggtgcccg gtgttgctgg accagacgga ccgggagtgg ccggtgtaac cggctggggc4358881ccaggcgatg gcgccggtgc cggagccggc tgcgggtgtg gagcgggagc tggggtaacg4358941ggcgtggccg gggttgccgg tgtggccggg gcgaccgggg gggtgaccgg cgtgatcggg4359001gttggctcgc ctggtgtgcc cggtttgacc ggggtcaccg gggtgaccgg cttgcccggg4359061gtcaccggcg tgacgggagt gccgggcgtt ggtgtgatcg gagttaccgg cgctcccggg4359121atgggtgtga ttggggttcc cggggtgatc ggggttcccg gggtgatcgg ggttcccggt4359181gtgcccggtg tgcccgggga tggcacgacc agggtaggca cgtctggggg tggcggcgac4359241ttctgctgaa gcaaatcctc gagtgcgttc ttcggaggtt tccaattctt ggattccagc4359301acccgctcag cggtctcggc gaccagactg acattggccc catgcgtcgc cgtgaccaat4359361gaattgatgg cggtatggcg ctcatcagca tccaggctag ggtcattctc caggatatcg4359421atctcccgtt gagcgccatc cacattattg ccgatatcgg atttagcttg ctcaatcaac4359481ccggcaatat gcctgtgcca ggtaatcacc gtggcgagat aatcctgcag cgtcatcaat4359541tgattgatgt ttgcacccag ggcgccgttg gcagcattgg cggcgccgcc ggaccatagg4359601ccgccttcga agacgtggcc tttctgctgg cggcaggtgt ccaatacatc ggtgaccctt4359661 tgcaaaacct ggctatattc ctgggcccgg tcatagaaag tgtcttcatc ggcttc

SEQ ID NO:2-A panCD region of Mycobacterium tuberculosis H37Rv, deleted in the ΔpanCD strain of Example 2.GGTCTAGCAGCTCGCCCGCGTTTTCGGGCACAAATGCCGGATCGTGGCCCATGTCGATCGGTTTGTTGTAAGCGTCGACAAACACGATCCGCGGCTGGTATGTGCGGGCCCGGGCGTCGTCCATCGTCGCGTACGCAATCAGAATCACCAGATCCCCCGGATGCACCAAGTGCGCGGCGGCACCGTTGATGCCAATCACACCACTGCCGCGTTCGCCGGTGATCGCGTAGGTGACCAGTCGAGCACCGTTGTCGATATCGACGATGGTTACCTGTTCGCCTTCCAGCAGGTCGGCGGCGTCCATCAAGTCGGCATCGATGGTCACCGAGCCGACGTAGTGCAGGTCGGCGCAGGTCACCGTGGCGCGGTGGATCTTCGACTTCAGCATCGTCCGTAACATCAGTTTCTCCAATGTGATTCGAGGATTGCCCGGTATCCGTCCGGGCGGTCGGTGCCGGCGAAAGTTCCGATTTCAATCGCAATGTTGTCCAGCAGCCTGGTGGTGCCAAGCCGGGCAGCAACCAGCAGCCGACCGGAACCGTTGAGCGGCATCCGGGCCAAGCCCGATATCGCGCAGCTCCAGGTAGTCGACCGCCACGCCGGGTGCAGCGTCGAGCACCGCACGGGCGGCATCCAGCGCGGCGTGCGCGCCAGCCGTTGCCGCATGCGCTGCGGCCGTTAGCGCCGCCGAGAGCGCGACGGCCGCCGCACGCTGGGCCGGGTCCAGGTAGCGGTTGCGCGACGACATCGCCAGCCCGTCGGCTTCGCGCACGGTCGGCACGCCGACCACCGCGACATCGAGGTTGAAGTCCGCGACCAGCTGCCGGATCAGCACCAGCTGCTGGTAGTCCTTCTCACCGAAGAACACCCGATCCGGGCGCACGATCTGCAGCAGCTTTAGCACGACCGTCAGCACGCCGGCGAAATGGGTTGGCCGCGGGCCGCCCTCGAGTTCGGCGGCCAACGGACCGGGTTGCACGGTGGTGCGCAGGCCGTCGGGATACATCGCCGCGGTAGTTGGCGTGAAAGCGATTTCCACGCCTTCGGCCCGCAGTTGCGCCAGGTCGTCGTCCGGGTGCGGGGATAGGCGTCGAGATCTTCCCCGGCACCGAATTGCATCGGGTTGACGAAGATCGACACGACGACGACCGATCCGGGCACCCGCTTGGCCGCACGCACCAACGCGAGGTGGCCTTCGTGCAGCGCACCCATAGTAGGCACCAACATCACTCGCCGGCCGGTGAGTCGCAGTGCGCGACTGACATCGGCGACATCCCCCGGTGCCGAGTACACATTGASEQ ID NO:3-A nadBC region of Mycobacterium tuberculosis H37Rv. Deleted in ΔnadBC strain of  Example 2.AACGGGCGATGAGCCGGGACGCGTCGATGTACCGCGCCGCCGCCGGGCTGCACCGGCTGTGCGACAGCCTATCCGGAGCACAGGTTCGCGACGTGGCTTGTCGCCGCGATTTCGAGGACGTGGCGCTCACGCTGGTCGCGCAGAGCGTGACCGCCGCCGCCTTGGCCCGCACCGAAAGCCGTGGCTGCCATCATCGCGCGGAGTACCCGTGCACCGTGCCGGAGCAGGCAGGCAGCATCGTGGTCCGGGGAGCCGACGACGCAAATGCGGTGTGTGTCCAGGCGCTAGTGGCGGTGTGCTGATGGGGTTATCCGACTGGGAGCTGGCTGCGGCTCGAGCAGCAATCGCGCGTGGGTCGACGAGGACCTCCGGTACGGCCCGGATGTCACCACATTGGCGACGGTGCCTGCCAGTGCGACGACCACCGCATCGCTGGTGACCCGGGAGGCCGGTGTGGTTGCCGGATTGGATGTCGCGCTGCTGACGCTGAACGAAGTCCTGGGCACCAACGGTTATCGGGTGCCGACCGCGTCGAGGACGGCGCCCGGGTGCCGCCGGGAGAGGCACTTATGACGCTGGAAGCCCAAACGCGCGGAATTGTTGACCGCCGAGCGCACCATGTTGAACCTGGTCGGTCACCTGTCGGGAATCGCCACCGCGACGGCCGCGTGGGTCGATGCTGTGCGCGGGACCAAAGCGAAAATCCGCGATACCCGTAAGACGCTGCCCGGCCTGCGCGCGCTGCAAAAA TACGCGGTGCGTACCGGTGGSEQ ID NO:4-A lysA sequence deleted in ΔlysA strainsGTGAACGAGCTGCTGCACTTAGCGCCGAATGTGTGGCCGCGCAATACTACTCGCGATGAAGTCGGTGTGGTCTGCATCGCAGGAATTCCACTGACGCAGCTCGCCCAGGAGTACGGGACCCCGCTGTTCGTCATCGACGAGGACGACTTTCGCTCGCGCTCGAGAAACCGCCGCGGCCTTTGGAAGTGGGGCGAACGTGCACTATGCCGCCAAGGCGTTCCTGTGCAGCGAAGTAGCCCGGTGGATCAGCGAAGAAGGGCTCTGTCTGGACGTTTGCACCGGTGGGGAGTTGGCGGTCGCGCTGCACGCTAGCTTTCCGCCCGAGCGAATTACCTTGCACGGCAACAACAAATCGGTCTCAGAGTTGACCGCTGCGGTCAAAGCCGGAGTCGGCCATATTGTCGTCGATTCGATGACCGAGATCGAGCGCCTCGACGCCATCGCGGGCGAGGCCGGAATCGTCCAGGATGTCCTGGTGCGTCTCACCGTCGGTGTCGAGGCGCACACCCACGAGTTCATCTCCACCGCGCACGAGACGCGTCAGCCACATCGGTTCGCAGATCTTCGACGTGGACGGCTTCGAACTCGCCGCGCACCGTGTCATCGGCCTGCTACGCGACGTCGTCGGCGAGTTCGGTCCCGAAAAGACGGCACAGATCGCGACCGTCGATCTCGGTGGCGGCTTGGGCATCTCGTATTTTGCCGTCCGACGACCCACCGCCGATAGCCGAGCTCGCGGCCAAGCTGGGTACCATCGTGAGCGACGAGTCAACGGCCGTGGGGCTGCCGACGCCCAAGCTCGTTGTGGAGCCCGGACGCGCCATCGCCGGACCGGGCACCATCACGTGTATGAGGTCGGCACCGTTAAGGACGTCGATGTCAGCGCCACAGCGCATCGACGTTACGTCAGTGTCGACGGCGGCATGAGCGACAACATCCGCACCGCGCTCTACGGCGCGCAGTATGACGTCCGGCTGGTGTCTCGAGTCAGCGACGCCCCGCCGGTACCGGCCCGTCTGGTCGGAAAGCACTGCGAAAGTGGCGATATCATCGTGCGGGACACCTGGGTGCCCGACGATATTCGGCCCGGCGATCTGGTTGCGGTTGCCGCCACCGGCGCTTACTGCTATTCGCTGTCGAGTCGTTACAACATGGTCGGCCGTCCCGCTGTGGTAGCGGTGCACGCGGGCAACGCTCGCCTGGTCCTGCGTCGGGAGACGGTCGACGATTTGCTGAGTTTGGAAGTGAGG TGASEQ ID NO:5-primer TH201 GGGGGCGCACCTCAAACC SEQ ID NO:6-primer TH202ATGTGCCAATCGTCGACCAGAA SEQ ID NO:7-primer TH203 CACCCAGCCGCCCGGATSEQ ID NO:8-primer TH204 TTCCTGATGCCGCCGTCTGA SEQ ID NO:9-primer Pan1GTGCAGCGCCATCTCTCA SEQ ID NO:10-primer Pan2 GTTCACCGGGATGGAACGSEQ ID NO:11-primer Pan3 CCCGGCTCGGTGTGGGAT SEQ ID NO:12-primer Pan4GCGCGGTATGCCCGGTAG

1. A method for inoculating an immunocompromised mammal against Mycobacterium tuberculosis, wherein the mammal does not have severe combined immune deficiency but is deficient in CD4⁺ lymphocytes or in CD8⁺ lymphocytes, the method comprising administering to the immunocompromised mammal an amount of an attenuated M. tuberculosis or M. bovis mycobacterium effective to confer protection against Mycobacterium tuberculosis in the mammal, wherein the attenuated mycobacterium has (i) a deletion of RD1 and is auxotrophic for pantothenate, or (ii) is auxotrophic for both lysine and pantothenate.
 2. The method of claim 1, wherein the attenuated mycobacterium is an M. tuberculosis.
 3. The method of claim 1, wherein the attenuated mycobacterium is an M. bovis.
 4. The method of claim 1, wherein the mammal is a human.
 5. The method of claim 1, wherein the RD1 deletion is a ΔpanCD deletion.
 6. The method of claim 1, wherein the mammal is deficient in CD8⁺ lymphocytes.
 7. The method of claim 1, wherein the mammal is deficient in CD4⁺ lymphocytes.
 8. The method of claim 1, wherein the attenuated mycobacterium has a deletion of RD1 and is auxotrophic for pantothenate.
 9. The method of claim 1, wherein the attenuated mycobacterium is auxotrophic for both lysine and pantothenate. 